Stable and soluble antibodies inhibiting tnf alpha

ABSTRACT

The present invention relates to particularly stable and soluble scFv antibodies and Fab fragments specific for TNF, which comprise specific light chain and heavy chain sequences that are optimized for stability, solubility, in vitro and in vivo binding of TNF, and low immunogenicity. Said antibodies are designed for the diagnosis and/or treatment of TNF-mediated disorders. The nucleic acids, vectors and host cells for expression of the recombinant antibodies of the invention, methods for isolating them and the use of said antibodies in medicine are also disclosed.

RELATED INFORMATION

The present application is a divisional of U.S. application Ser. No.13/000,345 filed Dec. 20, 2010 (now allowed) which claims priority fromPCT/CH2009/000219, filed Jun. 25, 2009, which claims priority to61/155,041 filed Feb. 24, 2009; 61/075,956 filed Jun. 26, 2008;61/075,640, 61/075,697, and 61/075,692 all filed Jun. 25, 2008.

BACKGROUND OF THE INVENTION

Tumour necrosis factor alpha (TNFα, also known as cachectin), is anaturally occurring mammalian cytokine produced by numerous cell types,including monocytes and macrophages in response to endotoxin or otherstimuli. TNFα is a major mediator of inflammatory, immunological, andpathophysiological reactions (Grell, M., et al. (1995) Cell, 83:793-802).

Soluble TNFα is formed by the cleavage of a precursor transmembraneprotein (Kriegler, et al. (1988) Cell 53: 45-53), and the secreted 17kDa polypeptides assemble to soluble homotrimer complexes (Smith, et al.(1987), J. Biol. Chem. 262: 6951-6954; for reviews of TNFα, see Butler,et al. (1986), Nature 320:584; Old (1986), Science 230: 630). Thesecomplexes then bind to receptors found on a variety of cells. Bindingproduces an array of pro-inflammatory effects, including (i) release ofother pro-inflammatory cytokines such as interleukin IL-6, IL-8, andIL-1, (ii) release of matrix metalloproteinases and (iii) up regulationof the expression of endothelial adhesion molecules, further amplifyingthe inflammatory and immune cascade by attracting leukocytes intoextravascular tissues.

A large number of disorders are associated with elevated levels of TNFα,many of them of significant medical importance. TNFα has been shown tobe up-regulated in a number of human diseases, including chronicdiseases such as rheumatoid arthritis (RA), inflammatory bowel disordersincluding Crohn's disease and ulcerative colitis, sepsis, congestiveheart failure, asthma bronchiale and multiple sclerosis. Mice transgenicfor human TNFα produce high levels of TNFα constitutively and develop aspontaneous, destructive polyarthritis resembling RA (Keffer et al.1991, EMBO J., 10, 4025-4031). TNFα is therefore referred to as apro-inflammatory cytokine.

TNFα is now well established as key in the pathogenesis of RA, which isa chronic, progressive and debilitating disease characterised bypolyarticular joint inflammation and destruction, with systemic symptomsof fever and malaise and fatigue. RA also leads to chronic synovialinflammation, with frequent progression to articular cartilage and bonedestruction. Increased levels of TNFα are found in both the synovialfluid and peripheral blood of patients suffering from RA. When TNFαblocking agents are administered to patients suffering from RA, theyreduce inflammation, improve symptoms and retard joint damage (McKown etal. (1999), Arthritis Rheum. 42:1204-1208).

Physiologically, TNFα is also associated with protection from particularinfections (Cerami. et al. (1988), Immunol. Today 9:28). TNFα isreleased by macrophages that have been activated by lipopolysaccharidesof Gram-negative bacteria. As such, TNFα appears to be an endogenousmediator of central importance involved in the development andpathogenesis of endotoxic shock associated with bacterial sepsis(Michie, et al. (1989), Br. J. Surg. 76:670-671.; Debets. et al. (1989),Second Vienna Shock Forum, p. 463-466; Simpson, et al. (1989) Crit. CareClin. 5: 27-47; Waage et al. (1987). Lancet 1: 355-357; Hammerle. et al.(1989) Second Vienna Shock Forum p. 715-718; Debets. et al. (1989),Crit. Care Med. 17:489-497; Calandra. et al. (1990), J. Infect. Dis.161:982-987; Revhaug et al. (1988), Arch. Surg. 123:162-170).

As with other organ systems, TNFα has also been shown to play a key rolein the central nervous system, in particular in inflammatory andautoimmune disorders of the nervous system, including multiplesclerosis, Guillain-Barre syndrome and myasthenia gravis, and indegenerative disorders of the nervous system, including Alzheimer'sdisease, Parkinson's disease and Huntington's disease. TNFα is alsoinvolved in disorders of related systems of the retina and of muscle,including optic neuritis, macular degeneration, diabetic retinopathy,dermatomyositis, amyotrophic lateral sclerosis, and muscular dystrophy,as well as in injuries to the nervous system, including traumatic braininjury, acute spinal cord injury, and stroke.

Hepatitis is another TNFα-related inflammatory disorder which amongother triggers can be caused by viral infections, includingEpstein-Barr, cytomegalovirus, and hepatitis A-E viruses. Hepatitiscauses acute liver inflammation in the portal and lobular region,followed by fibrosis and tumor progression. TNFα can also mediatecachexia in cancer, which causes most cancer morbidity and mortality(Tisdale M. J. (2004), Langenbecks Arch Surg. 389:299-305).

The key role played by TNFα in inflammation, cellular immune responsesand the pathology of many diseases has led to the search for antagonistsof TNFα. One class of TNFα antagonists designed for the treatment ofTNFα-mediated diseases are antibodies or antibody fragments thatspecifically bind TNFα and thereby block its function. The use ofanti-TNFα antibodies has shown that a blockade of TNFα can reverseeffects attributed to TNFα including decreases in IL-1, GM-CSF, IL-6,IL-8, adhesion molecules and tissue destruction (Feldmann et al. (1997),Adv. Immunol. 1997:283-350). Among the specific inhibitors of TNFα thathave recently become commercially available include a monoclonal,chimeric mouse-human antibody directed against TNFα (infliximab,Remicade™; Centocor Corporation/Johnson & Johnson) has demonstratedclinical efficacy in the treatment of RA and Crohn's disease. Allmarketed inhibitors of TNFα are administered intravenously orsubcutaneously in weekly or longer intervals as bolus injections,resulting in high starting concentrations that are steadily decreasinguntil the next injection. Their volume of distribution is limited.

Despite these advances, there remains a need for new and effective formsof antibodies or other immunobinders for the treatment forTNFα-associated disorders such as RA. In particular, there is an urgentneed for immunobinders with optimal functional properties for theeffective and continuous treatment of arthritis and other TNFα-mediateddisorders which allow for more flexible administration and formulationand have an improved tissue penetration and thereby an increased volumeof distribution.

SUMMARY OF THE INVENTION

Hence, it is a general object of the invention to provide a stable andsoluble antibody or other immunobinder, which specifically binds TNFα invitro and in vivo. In a preferred embodiment said immunobinder is anscFv antibody or Fab fragment.

The present invention provides stable and soluble scFv antibodies andFab fragments specific for TNFα, which comprise specific light chain andheavy chain sequences that are optimized for stability, solubility, invitro and in vivo binding of TNFα, and low immunogenicity. Saidantibodies are designed for the diagnosis and/or treatment ofTNFα-mediated disorders. The nucleic acids, vectors and host cells forexpression of the recombinant antibodies of the invention, methods forisolating them and the use of said antibodies in medicine are alsodisclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B depict the relative ability of the supernatants from44 anti-TNF RabMab hybridomas in binding TNFα (Biacore assay) andneutralising its activity (L929 assay).

FIG. 2 depicts the ability of 20 anti-TNF RabMab single-chain antibodiesand 7 humanized anti-TNF single-chain antibodies in selectively bindingTNFα (ELISA secretion assay, please note that for this assay supernatantfrom bacterial culture was used, which was not normalized forsingle-chain antibody content).

FIG. 3 depicts the binding kinetics (FIG. 3A) of EP43max and the bindingkinetics (FIG. 3B) of EP34max to human TNF alpha.

FIG. 4 depicts the potency of EP43max (open squares) the potency (closedcircles) of ESBA105. The EC50 of EP43max is 1 ng/ml and the EC50 ofESBA105 is 6.5 ng/ml

FIG. 5 depicts the performance of EP43max, EP6max and EP19max in athermal unfolding assay (FTIR).

FIG. 6 depicts the thermal denaturation curves of EP43max andderivatives thereof as compared by FTIR analysis.

FIG. 7 depicts the comparison of EP43 max (FIG. 7A) and its EP43minmaxvariant (FIG. 7B) in a thermal stress test.

FIG. 8 depicts the CDR H1 definition used herein for grafting antigenbinding sites from rabbit monoclonal antibodies into the highly solubleand stable human antibody frameworks.

FIG. 9A illustrates the potency of Epi34max and Adalimumab to blockcytotoxic activity of 1000 pg/ml recombinant human TNFalpha (murine L929cells). The IC₅₀ for Ep34max and Adalimumab was determined to be 1.03ng/ml and 8.46 ng/ml, respectively. FIG. 9B illustrates the potency ofAdalimumab and Ep34max to block cytotoxic activity of 10 pg/mlrecombinant human TNFalpha (human Kym-1 cells). The C₅₀ for Infliximaband Ep34max (791) was determined to be 66.2 ng/ml and 0.69 ng/mlrespectively.

FIG. 10A illustrates the potency of Epi34max and Infliximab to blockcytotoxic activity of 1000 pg/ml recombinant human TNFalpha (murine L929cells). The IC₅₀ for Ep34max and Infliximab was determined to be 1.04ng/ml and 13.9 ng/m, respectively.

FIG. 10B illustrates the potency of Infliximab and Ep34max (791) toblock cytotoxic activity of 10 pg/ml recombinant human TNFalpha (humanKym-1 cells). The IC₅₀ for Infliximab and Ep34max was determined to be14.8 ng/ml and 0.63 ng/ml respectively.

FIG. 11 illustrates the BioATR FT-IR thermal denaturation profile fittedfrom Fourier transformed infrared spectra in the amide I band region ofEp34 max in comparison to ESBA903. V50 for ESBA903 was 71.12 and forEP34max 71.50; the slope or ESBA903 2.481 and 2.540 for EP34max.

FIG. 12 illustrates the DSC thermal unfolding curves of Ep34max andESBA903 scFv antibodies. Tm of EP 34max is 78.11° C. and Tm for ESBA903is 76.19° C.

DETAILED DESCRIPTION OF THE INVENTION

It is a general object of the invention to provide stable and solubleimmunobiner which specifically binds TNFα in vitro and in vivo. In apreferred embodiment said antibody derivative is a scFv antibody or Fabfragment. The immunobinders of the invention preferably comprise a lightand/or heavy chain

DEFINITIONS

In order that the present invention may be more readily understood,certain terms will be defined as follows. Additional definitions are setforth throughout the detailed description.

The term “antibody” as used herein is a synonym for “immunoglobulin.”Antibodies according to the present invention may be wholeimmunoglobulins or fragments thereof, comprising at least one variabledomain of an immunoglobulin, such as single variable domains, Fv (SkerraA. and Pluckthun, A. (1988) Science 240:1038-41), scFv (Bird, R. E. etal. (1988) Science 242:423-26; Huston, J. S. et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-83), Fab, (Fab′)₂ or other fragments well knownto a person skilled in the art.

The term “CDR” refers to one of the six hypervariable regions within thevariable domains of an antibody that mainly contribute to antigenbinding. One of the most commonly used definitions for the six CDRs wasprovided by Kabat E. A. et al., (1991) Sequences of proteins ofimmunological interest. NIH Publication 91-3242). As used herein,Kabat's definition of CDRs only apply for CDR1, CDR2 and CDR3 of thelight chain variable domain (CDR L1, CDR L2, CDR L3, or L1, L2, L3), aswell as for CDR2 and CDR3 of the heavy chain variable domain (CDR H2,CDR H3, or H2, H3). CDR1 of the heavy chain variable domain (CDR H1 orH1), however, as used herein is defined by the following residues (Kabatnumbering): It starts with position 26 and ends prior to position 36.This is basically a fusion of CDR H1 as differently defined by Kabat andChotia (see also FIG. 8 for illustration).

The term “antibody framework” as used herein refers to the part of thevariable domain, either VL or VH, which serves as a scaffold for theantigen binding loops (CDRs) of this variable domain. In essence it isthe variable domain without the CDRs.

The term “single chain antibody”, “single chain Fv” or “scFv” isintended to refer to a molecule comprising an antibody heavy chainvariable domain (or region; V_(H)) and an antibody light chain variabledomain (or region; V_(L)) connected by a linker. Such scFv molecules canhave the general structures: NH₂-V_(L)-linker-V_(H)-COOH orNH₂-V_(H)-linker-V_(L)-COOH.

The term “immunobinder” refers to a molecule that contains all or a partof the antigen binding site of an antibody, e.g., all or part of theheavy and/or light chain variable domain, such that the immunobinderspecifically recognizes a target antigen. Non-limiting examples ofimmunobinders include full-length immunoglobulin molecules and scFvs, aswell as antibody fragments, including but not limited to (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fab′ fragment; (iv) a Fd fragment consisting of theV_(H) and C_(H)1 domains; (v) a Fv fragment consisting of the V_(L) andV_(H) domains of a single arm of an antibody, (vi) a single domainantibody such as a Dab fragment which consists of a V_(H) or V_(L)domain, a Camelid, or a Shark antibody (e.g., shark Ig-NARsNanobodies®); and (vii) a nanobody, a heavy chain region containing thevariable domain and two constant domains.

The numbering systems as used herein to identify amino acid residuepositions in antibody heavy and light chain variable regions correspondsto the one as defined by A. Honegger, J. Mol. Biol. 309 (2001) 657-670(the AHo system). Conversion tables between the AHo system and the mostcommonly used system as defined by Kabat et al. (Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) are provided in A. Honegger, J. Mol. Biol. 309 (2001) 657-670.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds (e.g.,TNF). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. See,e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996).

The terms “specific binding,” “selective binding,” “selectively binds,”and “specifically binds,” refer to antibody binding to an epitope on apredetermined antigen.

Typically, the antibody binds with an affinity (K_(D)) of approximatelyless than 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower.

The term “K_(D),” refers to the dissociation equilibrium constant of aparticular antibody-antigen interaction. Typically, the antibodies ofthe invention bind to TNF with a dissociation equilibrium constant(K_(D)) of less than approximately 10⁻⁷ M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower, for example, asdetermined using surface plasmon resonance (SPR) technology in a BIACOREinstrument.

As used herein, “identity” refers to the sequence matching between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of the two DNAmolecules is occupied by adenine, or a position in each of twopolypeptides is occupied by a lysine), then the respective molecules areidentical at that position. The “percentage identity” between twosequences is a function of the number of matching positions shared bythe two sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched, thenthe two sequences have 60% identity. By way of example, the DNAsequences CTGACT and CAGGTT share 50% identity (3 of the 6 totalpositions are matched). Generally, a comparison is made when twosequences are aligned to give maximum identity. Such alignment can beprovided using, for instance, the method of Needleman et al. (1970) J.Mol. Biol. 48: 443-453, implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.). The percent identity betweentwo amino acid sequences can also be determined using the algorithm ofE. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) whichhas been incorporated into the ALIGN program (version 2.0), using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. In addition, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch (J. Mol.Biol. 48:444-453 (1970)) algorithm which has been incorporated into theGAP program in the GCG software package (Accelrys, Inc., San Diego,Calif.), using either a Blossum 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6.

“Similar” sequences are those which, when aligned, share identical andsimilar amino acid residues, where similar residues are conservativesubstitutions for corresponding amino acid residues in an alignedreference sequence. In this regard, a “conservative substitution” of aresidue in a reference sequence is a substitution by a residue that isphysically or functionally similar to the corresponding referenceresidue, e.g., that has a similar size, shape, electric charge, chemicalproperties, including the ability to form covalent or hydrogen bonds, orthe like. Thus, a “conservative substitution modified” sequence is onethat differs from a reference sequence or a wild-type sequence in thatone or more conservative substitutions are present. The “percentagesimilarity” between two sequences is a function of the number ofpositions that contain matching residues or conservative substitutionsshared by the two sequences divided by the number of positionscompared×100. For instance, if 6 of 10 of the positions in two sequencesare matched and 2 of 10 positions contain conservative substitutions,then the two sequences have 80% positive similarity.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not negativelyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative sequence modificationsinclude nucleotide and amino acid substitutions, additions anddeletions. For example, modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions includeones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue in a human anti-VEGF antibody is preferably replaced withanother amino acid residue from the same side chain family. Methods ofidentifying nucleotide and amino acid conservative substitutions whichdo not eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997))

“Amino acid consensus sequence” as used herein refers to an amino acidsequence that can be generated using a matrix of at least two, andpreferably more, aligned amino acid sequences, and allowing for gaps inthe alignment, such that it is possible to determine the most frequentamino acid residue at each position. The consensus sequence is thatsequence which comprises the amino acids which are most frequentlyrepresented at each position. In the event that two or more amino acidsare equally represented at a single position, the consensus sequenceincludes both or all of those amino acids.

The amino acid sequence of a protein can be analyzed at various levels.For example, conservation or variability can be exhibited at the singleresidue level, multiple residue level, multiple residue with gaps etc.Residues can exhibit conservation of the identical residue or can beconserved at the class level. Examples of amino acid classes includepolar but uncharged R groups (Serine, Threonine, Asparagine andGlutamine); positively charged R groups (Lysine, Arginine, andHistidine); negatively charged R groups (Glutamic acid and Asparticacid); hydrophobic R groups (Alanine, Isoleucine, Leucine, Methionine,Phenylalanine, Tryptophan, Valine and Tyrosine); and special amino acids(Cysteine, Glycine and Proline). Other classes are known to one of skillin the art and may be defined using structural determinations or otherdata to assess substitutability. In that sense, a substitutable aminoacid can refer to any amino acid which can be substituted and maintainfunctional conservation at that position.

It will be recognized, however, that amino acids of the same class mayvary in degree by their biophysical properties. For example, it will berecognized that certain hydrophobic R groups (e.g., Alanine, Serine, orThreonine) are more hydrophilic (i.e., of higher hydrophilicity or lowerhydrophobicity) than other hydrophobic R groups (e.g., Valine orLeucine). Relative hydrophilicity or hydrophobicity can be determinedusing art-recognized methods (see, e.g., Rose et al., Science, 229:834-838 (1985) and Corvette et al., J. Mol. Biol., 195: 659-685 (1987)).

As used herein, when one amino acid sequence (e.g., a first V_(H) orV_(L) sequence) is aligned with one or more additional amino acidsequences (e.g., one or more VH or VL sequences in a database), an aminoacid position in one sequence (e.g., the first V_(H) or V_(L) sequence)can be compared to a “corresponding position” in the one or moreadditional amino acid sequences. As used herein, the “correspondingposition” represents the equivalent position in the sequence(s) beingcompared when the sequences are optimally aligned, i.e., when thesequences are aligned to achieve the highest percent identity or percentsimilarity.

“Chimeric” immunobinders as used herein have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies. Humanizedantibody as used herein is a subset of chimeric antibodies.

“Humanized antibodies” as used herein are immunobinders that have beensynthesized using recombinant DNA technology to circumvent immuneresponse to foreign antigens. Humanization is a well-establishedtechnique for reducing the immunogenicity of monoclonal antibodies ofxenogenic sources. A humanized antibody consists of humanized heavychain variable region, a humanized light chain variable region and fullyhuman constant domains. The humanization of a variable region involvesthe choice of an acceptor framework, typically a human acceptorframework, the extent of the CDRs from the donor immunobinder to beinserted into the variable domain acceptor framework and thesubstitution of residues from the donor framework into the acceptorframework. A general method for grafting CDRs into human acceptorframeworks has been disclosed by Winter in U.S. Pat. No. 5,225,539,which is hereby incorporated by reference in its entirety. U.S. Pat. No.6,407,213 the teachings of which are incorporated by reference in itsentirety, discloses a number of amino acid positions of the frameworkwhere a substitution from the donor immunobinder is preferred.

As used herein, the term “functional property” is a property of apolypeptide (e.g., an immunobinder) for which an improvement (e.g.,relative to a conventional polypeptide) is desirable and/or advantageousto one of skill in the art, e.g., in order to improve the manufacturingproperties or therapeutic efficacy of the polypeptide. In oneembodiment, the functional property is improved stability (e.g., thermalstability). In another embodiment, the functional property is improvedsolubility (e.g., under cellular conditions). In yet another embodiment,the functional property is non-aggregation. In still another embodiment,the functional property is an improvement in expression (e.g., in aprokaryotic cell). In yet another embodiment the functional property isan improvement in refolding yield following an inclusion bodypurification process. In certain embodiments, the functional property isnot an improvement in antigen binding affinity.

The term “nucleic acid molecule,” refers to DNA molecules and RNAmolecules.

A nucleic acid molecule may be single-stranded or double-stranded, butpreferably is double-stranded DNA. A nucleic acid is “operably linked”when it is placed into a functional relationship with another nucleicacid sequence. For instance, a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence.

The term “vector,” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.

The term “host cell” refers to a cell into which and expression vectorhas been introduced. Host cells can include bacterial, microbial, plantor animal cells. Bacteria, which are susceptible to transformation,include members of the enterobacteriaceae, such as strains ofEscherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis;Pneumococcus; Streptococcus, and Haemophilus influenzae. Suitablemicrobes include Saccharomyces cerevisiae and Pichia pastoris. Suitableanimal host cell lines include CHO (Chinese Hamster Ovary lines) and NS0cells.

The terms “treat,” “treating,” and “treatment,” refer to therapeutic orpreventative measures described herein. The methods of “treatment”employ administration to a subject, in need of such treatment, anantibody of the present invention, for example, a subject having aTNFα-mediated disorder or a subject who ultimately may acquire such adisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disorder or recurring disorder,or in order to prolong the survival of a subject beyond that expected inthe absence of such treatment.

The term “TNF-mediated disorder” or “TNF-mediated disease” refers to anydisorder, the onset, progression or the persistence of the symptoms ordisease states of which requires the participation of TNF. ExemplaryTNF-mediated disorders include, but are not limited to, chronic and/orautoimmune states of inflammation in general, immune mediatedinflammatory disorders in general, inflammatory CNS disease,inflammatory diseases affecting the eye, joint, skin, mucuous membranes,central nervous system, gastrointestinal tract, urinary tract or lung,states of uveitis in general, retinitis, HLA-B27+ uveitis, Behcet'sdisease, dry eye syndrome, glaucoma, Sjögren syndrome, diabetes mellitus(incl. diabetic neuropathy), insulin resistance, states of arthritis ingeneral, rheumatoid arthritis, osteoarthritis, reactive arthritis andReiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiplesclerosis, Guillain-Barre syndrome, myasthenia gravis, amyotrophiclateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidneydisease, cystitis, psoriasis (incl. psoriatic arthritis), hidradenitissuppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome(synovitis, acne, pustulosis, hyperostosis and osteitis), acne, Sweet'ssydrome, pemphigus, Crohn's disease (incl. extraintestinalmanifestastations), ulcerative colitis, asthma bronchiale,hypersensitivity pneumonitis, general allergies, allergic rhinitis,allergic sinusitis, chronic obstructive pulmonary disease (COPD), lungfibrosis, Wegener's granulomatosis, Kawasaki syndrome, Giant cellarteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graftversus host disease, host versus graft reactions, rejection episodesfollowing organ or bone marrow transplantation, sytemic and local statesof vasculitis in general, systemic and discoid lupus erythematodes,polymyositis and dermatomyositis, sclerodermia, pre-eclampsia, acute andchronic pancreatitis, viral hepatitis, alcoholic hepatitis, postsurgicalinflammation such as after eye surgery (e.g. cataract (eye lensreplacement) or glaucoma surgery), joint surgery (incl. arthroscopicsurgery), surgery at joint-related structures (e.g. ligaments), oraland/or dental surgery, minimally invasive cardiovascular procedures(e.g. PTCA, atherectomy, stent placement), laparoscopic and/orendoscopic intra-abdominal and gynecological procedures, endoscopicurological procedures (e.g. prostate surgery, ureteroscopy, cystoscopy,interstitial cystitis), or perioperative inflammation (prevention) ingeneral, Alzheimer disease, Parkinson's disease, Huntington's disease,Bell' palsy, Creutzfeld-Jakob disease. Cancer-related osteolysis,cancer-related inflammation, cancer-related pain, cancer-relatedcachexia, bone metastases, acute and chronic forms of pain, irrespectivewhether these are caused by central or peripheral effects of TNFα andwhether they are classified as inflammatory, nociceptive or neuropathicforms of pain, sciatica, low back pain, carpal tunnel syndrome, complexregional pain syndrome (CRPS), gout, postherpetic neuralgia,fibromyalgia, local pain states, chronic pain syndroms due to metastatictumor, dismenorrhea. Bacterial, viral or fungal sepsis, tuberculosis,AIDS, atherosclerosis, coronary artery disease, hypertension,dyslipidemia, heart insufficiency and chronic heart failure. The term“effective dose” or “effective dosage” refers to an amount sufficient toachieve or at least partially achieve the desired effect. The term“therapeutically effective dose” is defined as an amount sufficient tocure or at least partially arrest the disease and its complications in apatient already suffering from the disease. Amounts effective for thisuse will depend upon the severity of the disorder being treated and thegeneral state of the patient's own immune system.

The term “subject” refers to any human or non-human animal. For example,the methods and compositions of the present invention can be used totreat a subject with a TNF-mediated disorder.

The term “lagomorphs” refers to members of the taxonomic orderLagomorpha, comprising the families Leporidae (e.g. hares and rabbits),and the Ochotonidae (pikas). In a most preferred embodiment, thelagomorphs is a rabbit. The term “rabbit” as used herein refers to ananimal belonging to the family of the leporidae.

Different nomenclatures were used for the generated immunobinders. Theseare typically identified by a number (e.g. #34). In those cases where aprefix such as EP or Epi was used (e.g. EP 34 which is identical to Epi34 or to #34), the same immunobinder is thereby indicated.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending ofthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

Anti-TNFα Antibodies

In one aspect, the present invention provides immunobinders that bindTNFα and thus are suitable to block the function of TNFα in vivo. TheCDRs of these immunobinders are derived from rabbit anti-TNFα monoclonalantibodies as disclosed in U.S. Pat. No. 7,431,927. Rabbit antibodiesare known to have particularly high affinities. Moreover, the CDRsequences disclosed herein are natural sequences, which means that noaffinity maturation of the resulting immunobinders needs to beperformed. In a preferred embodiment, the immunobinder neutralizes TNFαin vivo.

In certain embodiments, the invention provides an immunobinder, whichspecifically binds TNFα, comprising at least one of a CDRH1, a CDRH2, aCDRH3, a CDRL1, a CDRL2, or a CDRL3 amino acid sequence. Exemplary CDRamino acid sequences for use in the immunobinders of the invention areset forth in SEQ ID Nos: 3-50 (Table 1). The CDRs set forth in SEQ IDNos: 3-50 can be grafted onto any suitable binding scaffold using anyart recognized methods (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.). The CDRs from different parentantibodies may be combined into one antibody to generate additionalantibody species. However, it is preferred that the immunobindersdisclosed herein are humanized, thus being suitable for therapeuticapplications.

Thus, in one embodiment, the invention provides an immunobinder whichspecifically binds human TNFα, the immunobinder comprising:

-   -   (i) a humanized heavy chain variable region (VH), the heavy        chain variable region comprising a human heavy chain variable        framework sequence and CDR H1, CDR H2 and CDR H3 sequences        stemming from a rabbit immunobinder; and/or    -   (ii) a humanized light chain variable region (VL), the light        chain variable region comprising a human light chain variable        framework sequence and CDR L1, CDR L2 and CDR L3 sequences        stemming from a rabbit immunobinder.

As known in the art, many rabbit VH chains have extra paired cysteinesrelative to the murine and human counterparts. In addition to theconserved disulfide bridge formed between cys22 and cys92, there is alsoa cys21-cys79 bridge as well as an interCDR S—S bridge formed betweenthe last residue of CDRH1 and the first residue of CDR H2 in some rabbitchains. Besides, pairs of cysteine residues are often found in theCDR-L3. Besides, many rabbit antibody CDRs do not belong to anypreviously known canonical structure. In particular the CDR-L3 is oftenmuch longer than the CDR-L3 of a human or murine counterpart.

Further to rabbits, the invention may be used for grafting CDRs of anylagomorph.

In the case of antibodies, the rabbit CDRs set forth in SEQ ID Nos: 3-50may be grafted into the framework regions of any antibody from anyspecies. However, it has previously been discovered that antibodies orantibody derivatives comprising the frameworks identified in the socalled “quality control” screen (WO0148017) are characterised by agenerally high stability and/or solubility and thus may also be usefulin the context of extracellular applications such as neutralizing humanTNFα. Moreover, it has further been discovered that one particularcombination of these VL (variable light chain) and VH (variable heavychain) soluble and stable frameworks is particularly suited toaccommodating rabbit CDRs. It was surprisingly found that upon graftinginto said framework or its derivatives, loop conformation of a largevariety of rabbit CDRs could be fully maintained, largely independent ofthe sequence of the donor framework. Moreover, said framework or itsderivatves containing different rabbit CDRs are well expressed and goodproduced contrary to the rabbit wild type single chains and still almostfully retain the affinity of the original donor rabbit antibodies.Accordingly, in one embodiment, the CDRs set forth in SEQ ID Nos: 3-50are grafted into the human antibody frameworks derived by “qualitycontrol” screening disclosed in EP1479694. The amino acid sequences ofexemplary frameworks for use in the invention are set forth in SEQ IDNos: 1 and 2 below.

SEQ ID No 1 Variable light chain of FW1.4EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50)WYQQKPGKAPKLLIY(X)_(n=3-50)VPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)_(n=3-50)FGQGTKLTVLG SEQ ID No 2Variable heavy chain of FW1.4EVQLVESGGGLVQPGGSLRLSCAAS(X)_(n=3-50)WVRQAPGKGLEWVS(X)_(n=3-50)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(X)_(n=3-50)WGQGTLVTVSS

X can be any naturally occurring amino acid. At least three and up to 50amino acids can be present. The CDRs are typically inserted into thesites where X is present.

In other embodiments, the invention provides an immunobinder, whichspecifically binds TNFα, comprising at least one of a VH or a VL aminoacid sequence. Exemplary VH or VL amino acid sequences for use in theimmunobinders of the invention are set forth in SEQ ID Nos: 51-111.

In certain embodiments, the invention further provides an immunobinder,which specifically binds TNFα, comprising an amino acid sequence withsubstantial similarity to an amino acid sequence set forth in SEQ IDNos: 51-111, and wherein the immunobinder retains or improves thedesired functional properties of the anti-TNFα immunobinder of theinvention. Exemplary percentage similarities include, but are notlimited to, about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity.

In certain embodiments, the invention further provides an immunobinder,which specifically binds TNFα, comprising an amino acid sequence withsubstantial identity to an amino acid sequence set forth in SEQ ID Nos:51-111, and wherein the immunobinder retains or improves the desiredfunctional properties of the anti-TNFα immunobinder of the invention.Exemplary percentage identities include, but are not limited to, about50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity.

In certain embodiments, the invention further provides an immunobinder,which specifically binds TNFα, comprising an amino acid sequence withconservative substitutions relative to an amino acid sequence set forthin SEQ ID Nos: 51-111, and wherein the immunobinder retains or improvesthe desired functional properties of the anti-TNFα immunobinder of theinvention.

In a most preferred embodiment, the immunobinder of the inventioncomprises at least one CDR sequence being at least 80%, more preferablyat least 85%, 90%, 95% or 100% identical to anyone of the SEQ ID Nos:3-50.

In a preferred embodiment of the invention, an immunobinder is providedcomprising at least one, preferably two, three, four, five or mostpreferably six CDRs of the group consisting of SEQ ID Nos 3-8.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos 9-14.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos: 15-20.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos: 21-26.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos: 27-32.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos: 33-38.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably, two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos: 39-44.

In another preferred embodiment of the invention, an immunobinder isprovided comprising at least one, preferably two, three, four, five ormost preferably six CDRs of the group consisting of SEQ ID Nos: 45-50.

The CDR sequences provided herein in SEQ ID Nos: 3-50 may furthercomprise substitutions. Preferably, the sequences have 3, morepreferably 2, and most preferably only one substitution(s). Saidsubstitutions are preferably such that the selective binding capacity ofthe immunobinder is not impaired but the affinity of the immunobinder isaltered, preferably enhanced.

TABLE 1 Rabmab Donor CDRs SEQ ID Rabmab Clone CDR Amino Acid SequenceNO: EP-43 CDR-H1 GFSLSSGAMS 3 CDR-H2 VIISSGATYYASWAKG 4 CDR-H3GGPDDSNSMGTFDP 5 CDR-L1 QASQSISDWLA 6 CDR-L2 GASRLAS 7 CDR-L3QQGWSDSYVDNL 8 EP-1 CDR-H1 GIDLSNDAIS 9 CDR-H2 YISDWSIRYYANWAQG 10CDR-H3 GAPGAGDNGI 11 CDR-L1 QSTESVYKNNYLA 12 CDR-L2 DASTLAS 13 CDR-L3AGYYRSGSGTANGS 14 EP-6 CDR-H1 GFSLSRYGVS 15 CDR-H2 TIGEAGRAYYANWARS 16CDR-H3 GEVFNNGWGAFNI 17 CDR-L1 QASESIYSGLA 18 CDR-L2 QASTLAS 19 CDR-L3QQGFGTSNVENP 20 EP-15 CDR-H1 GFSLSRYGVS 21 CDR-H2 AIGETGRAYYANWAKS 22CDR-H3 GEEFNNGWGAFNI 23 CDR-L1 QASENIYTSLA 24 CDR-L2 SASTLAS 25 CDR-L3QQGFATSNVENP 26 EP-19 CDR-H1 GFSLNSNEIS 27 CDR-H2 YIGNGGMTHYASWAKG 28CDR-H3 SVEYTDLYYLNI 29 CDR-L1 QASDNIYRGLA 30 CDR-L2 DASTLQS 31 CDR-L3LGVYGYSSDDGAA 32 EP-34 CDR-H1 GFTISRSYWIC 33 CDR-H2 CIYGDNDITPLYANWAKG34 CDR-H3 LGYADYAYDL 35 CDR-L1 QSSQSVYGNIWMA 36 CDR-L2 QASKLAS 37 CDR-L3QGNFNTGDRYA 38 EP-35 CDR-H1 GFSFSVGYWIC 39 CDR-H2 CIDAGTSGGTYYATWAKG 40CDR-H3 GVSSNGYYFKL 41 CDR-L1 QASQSISNLLA 42 CDR-L2 AASKLAS 43 CDR-L3QQGWSHTNVDNT 44 EP-42 CDR-H1 GIDLRNDAIS 45 CDR-H2 YISDWGIKYYASWVKG 46CDR-H3 GAPGAGDNGI 47 CDR-L1 QSTESVYKNNYLA 48 CDR-L2 DASTLAS 49 CDR-L3AGYYRSGFGTANG 50

In another embodiment, the invention provides antibodies that bind to anepitope on human TNFα as is recognized by a monoclonal antibodycontaining a set of CDRs (H1-H3, L1-L3; belonging to one Rabmab clone)as set forth in Table 1. Such antibodies can be identified based ontheir ability to cross-compete with an antibody of Table 1 in a standardTNF binding assay. The ability of a test antibody to inhibit the bindingof an antibody of Table 1 to human TNFα demonstrates that the testantibody can compete with the antibody of Table 1 for binding to humanTNFα and thus inyloves the same epitope on human TNFα as the antibody ofTable 1. In a preferred embodiment, the antibody that binds to the sameepitope on human TNFα as the antibodies set forth in Table 1 is a humanmonoclonal antibody. Such human monoclonal antibodies can be preparedand isolated as described herein.

In one embodiment, antibodies and antibody fragments of the presentinvention are single-chain antibodies (scFv) or Fab fragments. In thecase of scFv antibodies, a selected VL domain can be linked to aselected VH domain in either orientation by a flexible linker. Asuitable state of the art linker consists of repeated GGGGS (SEQ ID NO:122) amino acid sequences or variants thereof. In a preferred embodimentof the present invention a (GGGGS)₄ linker (SEQ ID No: 72) or itsderivative is used, but variants of 1-3 repeats are also possible(Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Otherlinkers that can be used for the present invention are described byAlfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061,Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al.(2001), Cancer Immunol. Immunother. 50:51-59. The arrangement can beeither NH₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH, with the formerorientation being the preferred one. In the case of Fab fragments,selected light chain variable domains VL are fused to the constantregion of a human Ig kappa chain, while the suitable heavy chainvariable domains VH are fused to the first (N-terminal) constant domainCH1 of a human IgG. At the C-terminus, an inter-chain disulfide bridgeis formed between the two constant domains.

The antibodies or antibody derivatives of the present invention can haveaffinities to human TNF with dissociation constants K_(d) in a range of1 fM-10 μM. In a preferred embodiment of the present invention the K_(d)is <1 nM. The affinity of an antibody for an antigen can be determinedexperimentally using a suitable method (Berzofsky et al.“Antibody-Antigen Interactions”, in Fundamental Immunology, Paul, W. E.,Ed, Raven Press: New York, N.Y. (1992); Kuby, J. Immunology, W.H.Freeman and Company: New York, N.Y.) and methods described therein.

Preferred antibodies include antibodies having a variable heavy (VH)and/or variable light (VL) chain region from among the following VH andVL sequences (CDR sequences underlined):

SEQ ID NO: 51 EP43min VH EVQLVESGGGLVQPGGSLRLSCAAS GFSLSSGAMSWVRQAPGKGLEWVS VIISSGATYY ASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGPDDSNSMGTFDP WGQGTLV TVSS SEQ ID NO: 52 EP43min VLEIVMTQSPSTLSASVGDRVIITC QASQSISDWLA WYQQKPGKAPKLLIY GASRLAS GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC QQGWSDSYVDNL FGQGTKLTVLG SEQ ID NO: 53EP43max VH EVQLVESGGGLVQPGGSLRLSCTVS GFSLSSGAMS WVRQAPGKGLEWVGVIISSGATYY ASWAKG RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR GGPDDSNSMGTFDPWGQGTLV TVSS SEQ ID NO: 54 EP43max VL EIVMTQSPSTLSASVGDRVIIKCQASQSISDWLA WYQQKPGKAPKLLIY GASRLAS GFP SRFSGSGSGAEFTLTISGLEPADFATYYCQQGWSDSYVDNL FGQGTKLTVLG SEQ ID NO: 55 EP43maxDHP VHEVQLVESGGGSVQPGGSLRLSCTVS GFSLSSGAMS WVRQAPGKGLEWVG VIISSGATYY ASWAKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCAR GGPDDSNSMGTFDP WGQGTSV TVSSSEQ ID NO: 56 EP43minmaxVL: T22K VL EIVMTQSPSTLSASVGDRVIIKC QASQSISDWLAWYQQKPGKAPKLLIY GASRLAS GVP SRFSGSGSGAEFTLTISSLQPDDFATYYC QQGWSDSYVDNLFGQGTKLTVLG SEQ ID NO: 57 EP43minmaxVL: V58F VL EIVMTQSPSTLSASVGDRVIITCQASQSISDWL AWYQQKPGKAPKLLIY GASRLAS GFP SRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSDSYVDNL FGQGTKLTVLG SEQ ID NO: 58 EP43minmaxVL: Q79E VLEIVMTQSPSTLSASVGDRVIITC QASQSISDWL AWYQQKPGKAPKLLIY GASRLAS GVPSRFSGSGSGAEFTLTISSLEPDDFATYYC QQGWSDSYVDNL FGQGTKLTVLG SEQ ID NO: 59EP43minmaxVL: D81A VL EIVMTQSPSTLSASVGDRVIITC QASQSISDWLAWYQQKPGKAPKLLIY GASRLAS GVP SRFSGSGSGAEFTLTISSLQPADFATYYC QQGWSDSYVDNLFGQGTKLTVLG SEQ ID NO: 60 EP1min VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFSNDAISWVRQAPGKGLEWVS YISDWSIRYY ANWAQG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAPGAGDNGI WGQGTLVTVSS NOTE: EP1min CDR-H1 does not match that of EP1maxSEQ ID NO: 61 EP1min VL EIVMTQSPSTLSASVGDRVIITC QSTESVYKNNYLAWYQQKPGKAPKLLIY DASTLAS G VPSRFSGSGSGAEFTLTISSLQPDDFATYYC AGYYRSGSGTANGSFGQGTKLTVLG SEQ ID NO: 62 EP1max VH EVQLVESGGGSVQPGGSLRLSCTVS GIDLSNDAISWVRQAPGKGLEWVA YISDWSIRYY ANWAQG RFTISKDTSKNTVYLQMNSLRAEDTATYYCARGAPGAGDNGI WGQGTTVTVSS SEQ ID NO: 63 EP1max VL EIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLA WYQQKPGKAPKLLIY DASTLAS G VPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGYYRSGSGTANGS FGQGTKLTVLG SEQ ID NO: 64 EP6min VHEVQLVESGGGLVQPGGSLRLSCAAS GFSLSRYGVS WVRQAPGKGLEWVS TIGEAGRAYY ANWARSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GEVFNNGWGAFNI WGQGTLVT VSSSEQ ID NO: 65 EP6min VL EIVMTQSPSTLSASVGDRVIITC QASESIYSGLAWYQQKPGKAPKLLIY QASTLAS GVP SRFSGSGSGAEFTLTISSLQPDDFATYYC QQGFGTSNVENPFGQGTKLTVLG SEQ ID NO: 66 EP6max VH EVQLVESGGGLVQPGGSLRLSCTVS GFSLSRYGVSWVRQAPGKGLEWVG TIGEAGRAYY ANWARS RSTISRDTSKNTVYLQMNSLRAEDTAVYYCARGEVFNNGWGAFNI WGQGTLVT VSS SEQ ID NO: 67 EP6max VLEIVMTQSPSTLSASVGDRVIITC QASESIYSGLA WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTDFTLAISSLQPDDFATYYC QQGFGTSNVENP FGQGTKLTVLG SEQ ID NO: 68EP15min VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFSRYGVS WVRQAPGKGLEWVSAIGETGRAYY ANWAK SRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GEEFNNGWGAFNIWGQGTLVT VSS SEQ ID NO: 69 EP15min VL EIVMTQSPSTLSASVGDRVIITCQASENIYTSLA WYQQKPGKAPKLLIY SASTLAS GVP SRFSGSGSGAEFTLTISSLQPDDFATYYCQQGFATSNVENP FGQGTKLTVLG SEQ ID NO: 70 EP15max VHEVQLVESGGGSVQPGGSLRLSCTVS GFSLSRYGVS WVRQAPGKGLEWVG AIGETGRAYY ANWAKSRSTISRDTSKNTVYLQMNSLRAEDTATYYCAR GEEFNNGWGAFNI WGQGTTVT VSSSEQ ID NO: 71 EP15max VL EIVMTQSPSTLSASVGDRVIITC QASENIYTSLAWYQQKPGKAPKLLIY SASTLAS GVP SRFSGSGSGTEFTLTISSLQPDDFATYYC QQGFATSNVENPFGQGTKLTVLG SEQ ID NO: 72 glycine-serine linker GGGGSGGGGSGGGGSGGGGSSEQ ID NO: 73 EP19maxmod VH EVQLVESGGGLVQPGGSLRLSCTVS GFSLNSNEISWVRQAPGKGLEWVG YIGNGGMTHY ASWAKG RFTISRDTSKNTVYLQMNSLRAEDTAVYYCASSVEYTDLYYLNI WGQGTLVTV SS SEQ ID NO: 74 EP19maxmod VLEIVMTQSPSTLSASVGDRVIITC QASDNIYRGLA WYQQKPGKAPKLLIY DASTLQS GVPSRFSGSGSGTQFTLTISSLQPDDFATYYC LGVYGYSSDDGAA FGQGTKLTVLG SEQ ID NO: 75EP19minmod VH EVQLVESGGGLVQPGGSLRLSCAAS GFSLNSNEIS WVRQAPGKGLEWVSYIGNGGMTHY ASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SVEYTDLYYLNIWGQGTLVTV SS SEQ ID NO: 76 EP19minmod VL EIVMTQSPSTLSASVGDRVIITCQASDNIYRGLA WYQQKPGKAPKLLIY DASTLQS GVP SRFSGSGSGAEFTLTISSLQPDDFATYYCLGVYGYSSDDGAA FGQGTKLTVLG SEQ ID NO: 77 EP34min VHEVQLVESGGGLVQPGGSLRLSCAAS GFTISRSYWIC WVRQAPGKGLEWVS CIYGDNDITPLYANWAKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAK LGYADYAYDL WGQGTLVT VSSSEQ ID NO: 78 EP34min VL EIVMTQSPSTLSASVGDRVIITC QSSQSVYGNIWMAWYQQKPGKAPKLLIY QASKLAS G VPSRFSGSGSGAEFTLTISSLQPDDFATYYC QGNFNTGDRYAFGQGTKLTVLG SEQ ID NO: 79 EP34max VH EVQLVESGGGLVQPGGSLRLSCTASGFTISRSYWIC WVRQAPGKGLEWVA CIYGDNDIT PLYANWAKGRFPVSTDTSKNTVYLQMNSLRAEDTAVYYCAR LGYADYAYDL WGQGTLVT VSS SEQ ID NO: 80EP34max VL EIVMTQSPSTLSASLGDRVIITC QSSQSVYGNIWMA WYQQKSGKAPKLLIY QASKLASG VPSRFSGSGSGAEFSLTISSLQPDDFATYYC QGNFNTGDRYA FGQGTKLTVLG SEQ ID NO: 81EP35min VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFSVGYWIC WVRQAPGKGLEWVSCIDAGTSGG TYYATWAK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GVSSNGYYFKL WGQGTLVTVSS SEQ ID NO: 82 EP35min VL EIVMTQSPSTLSASVGDRVIITC QASQSISNLLAWYQQKPGKAPKLLIY AASKLAS GVP SRFSGSGSGAEFTLTISSLQPDDFATYYC QQGWSHTNVDNTFGQGTKLTVLG SEQ ID NO: 83 EP35max VH EVQLVESGGGSVQPGGSLRLSCTASGFSFSVGYWIC WVRQAPGKGLEWVA CIDAGTSGG TYYATWAKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCAR GVSSNGYYFKL WGQGTTV TVSS SEQ ID NO: 84EP35max VL EIVMTQSPSTLSASVGDRVIITC QASQSISNLLA WYQQKPGKAPKLLIV AASKLASGVP SRFSGSGSGTEFTLTISSLQPDDFATYYC QQGWSHTNVDNT FGQGTKLTVLG SEQ ID NO: 85EP42min VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFRNDAIS WVRQAPGKGLEWVSYISDWGIKYY ASWVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GAPGAGDNGIWGQGTLVTVSS NOTE: EP42min CDR-H1 does not match that of EP42maxSEQ ID NO: 86 EP42min VL EIVMTQSPSTLSASVGDRVIITC QSTESVYKNNYLAWYQQKPGKAPKLLIY DASTLAS G VPSRFSGSGSGAEFTLTISSLQPDDFATYYC AGYYRSGFGTANGSFGQGTKLTVLG SEQ ID NO: 87 EP42max VH EVQLVESGGGSVQPGGSLRLSCTVSGIDLRNDAIS WVRQAPGKGLEWVS YISDWGIKYY ASWVKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCAR GAPGAGDNGI WGQGTTVTVSS SEQ ID NO: 88EP42max VL EIVMTQSP STLSASVGDRVIITC QSTESVYKNNYLA WYQQKPGKAPKLLIYDASTLAS GVPSRFSGS GSGTEFTLTISSLQPDDFATYYC AGYYRSGFGTANG SFGQGTKLTVLG

Production of Anti-TNF Antibodies

The present invention is based, at least in part, on the discovery thatthe highly soluble and stable human antibody frameworks identified by aQuality Control (QC) assay are particulary suitable frameworks foracommodating CDRs from other non-human animal species, for example,rabbit CDRs. In particular, the invention is based on the discovery thatthe light and heavy chain variable regions of particular human antibody(the so called, “FW 1.4” antibody) are particularly suitable asacceptors for CDRs from a variety of rabbit antibodies of differentbinding specificities. Although ESBATech's human single-chain frameworkFW1.4 clearly underperformed in the Quality Control assay and whenexpressed in HeLa cells when used together with its original CDRs (asdisclosed in WO03097697), it was surprisingly found that, when combinedwith other CDRs, such as rabbit CDRs, it gives rise to very stable,soluble and well producible single-chain antibodies. Furthermore,humanized immunobinders generated by the grafting of rabbit CDRs intothese highly compatible light and heavy frameworks consistently andreliably retain the binding properties of the rabbit antibodies fromwhich the donor CDRs are derived. Moreover, immunobinders generated bythe methods of the invention reliably exhibit superior functionalproperties such as solubility and stability. Accordingly, it is ageneral object of the invention to provide methods for grafting rabbitand other non-human CDRs, into the soluble and stable light chain and/orheavy chain human antibody frameworks of SEQ ID NO:1 (K127) and SEQ IDNO:2 (a43), respectively, thereby generating humanized antibodies withsuperior biophysical properties.

In a preferred embodiment, the framework comprises one or moresubstitutions in the heavy chain framework (VH) at a position from thegroup consisting of positions H24, H25, H56, H82, H84, H89 and H108 (AHonumbering system). Additionally or alternatively, the framework maycomprise a substitution in the light chain framework (VH) at positionL87 according to the AHo numbering system. The presence of saidsubstitutions have shown to provide an acceptor framework which almostfully retains the affinity of the original donor antibodies. In a morepreferred embodiment, the one or more of substitutions selected from thegroup consisting of: threonine (T) at position H24, valine (V) atpostion H25, glycine (G) or alanine (A) at position H56, lysine (K) atposition H82, threonine (T) at postion H84, valine (V) at position H89and arginine (R) at position H108 and threonine (T) at position L87according to the AHo numbering system are present in the frameworksequence.

Thus, in an even more preferred embodiment, the acceptor framework is

SEQ ID NO. 89: variable heavy chain framework of rFW1.4EVQLVESGGGLVQPGGSLRLSCTAS(X)_(n=3-50) WVRQAPGKGLEWVG(X)_(n=3-50)RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=3-50) WGQGTLV TVSSSEQ ID NO. 90: variable heavy chain framework of rFW1.4(V2)EVQLVESGGGLVQPGGSLRLSCTVS(X)_(n=3-50) WVRQAPGKGLEWVG(X)_(n=3-50)RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=3-50) WGQGTLVTVSSSEQ ID NO. 91: substituted variable light chain framework of FW1.4EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50) WYQQKPGKAPKLLIY(X)_(n=3-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=3-50) FGQGTKLTVLGSEQ ID NO. 92: framework of rFW1.4EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50) WYQQKPGKAPKLLIY(X)_(n=3-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=3-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS(X)_(n=3-50)WVRQAPGKGLEWVG(X)_(n=3-50) RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=3-50) WGQGTLVTVSSSEQ ID NO. 93: framework of rFW1.4(V2)EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50) WYQQKPGKAPKLLIY(X)_(n=3-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=3-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCTVS(X)_(n=3-50)WVRQAPGKGLEWVG(X)_(n=3-50) RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=3-50) WGQGTLVTVSS

X can be any naturally occurring amino acid; at least three and up to 50amino acids can be present. The CDRs are typically inserted into thesites where X is present.

The antibodies or antibody derivatives of the present invention may begenerated using routine techniques in the field of recombinant genetics.Knowing the sequences of the polypeptides, the cDNAs encoding them canbe generated by gene synthesis by methods well known in the art. ThesecDNAs can be cloned into suitable vector plasmids. Once the DNA encodinga VL and/or a VH domain are obtained, site directed mutagenesis, forexample by PCR using mutagenic primers, can be performed to obtainvarious derivatives. The best “starting” sequence can be chosendepending on the number of alterations desired in the VL and/or VHsequences. A preferred sequence is the TB-A sequences and itsderivatives, e.g. scFv sequences or Fab fusion peptide sequences, may bechosen as templates for PCR driven mutagenesis and/or cloning.

Methods for incorporating or grafting CDRs into framework regionsinclude those set forth in, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al, as well as those disclosed in U.S.Provisional Application Ser. Nos. 61/075,697 and 61/155,041, entitled“Humanization of Rabbit Antibodies Using Universal Antibody Frameworks,”filed on Jun. 25, 2008 and on Feb. 4, 2009, respectively.

Standard cloning and mutagenesis techniques well known to the personskilled in the art can be used to attach linkers, shuffle domains orconstruct fusions for the production of Fab fragments. Basic protocolsdisclosing the general methods of this invention are described inMolecular Cloning, A Laboratory Manual (Sambrook & Russell, 3^(rd) ed.2001) and in Current Protocols in Molecular Biology (Ausubel et al.,1999).

The DNA sequence harboring a gene encoding a scFv polypeptide, or in thecase of Fab fragments, encoding either two separate genes or abi-cistronic operon comprising the two genes for the VL-Cκ and theVH-CH1 fusions are cloned in a suitable expression vector, preferablyone with an inducible promoter. Care must be taken that in front of eachgene an appropriate ribosome binding site is present that ensurestranslation. It is to be understood that the antibodies of the presentinvention comprise the disclosed sequences rather than they consist ofthem. For example, cloning strategies may require that a construct ismade from which an antibody with one or a few additional residues at theN-terminal end are present. Specifically, the methionine derived fromthe start codon may be present in the final protein in cases where ithas not been cleaved posttranslationally. Most of the constructs forscFv antibodies give rise to an additional alanine at the N-terminalend. In a preferred embodiment of the present invention, an expressionvector for periplasmic expression in E. coli is chosen (Krebber, 1997).Said vector comprises a promoter in front of a cleavable signalsequence. The coding sequence for the antibody peptide is then fused inframe to the cleavable signal sequence. This allows the targeting of theexpressed polypeptide to the bacterial periplasm where the signalsequence is cleaved. The antibody is then folded. In the case of the Fabfragments, both the VL-Cκ and the VH-CH1 fusions peptides must be linkedto an export signal. The covalent S—S bond is formed at the C-terminalcysteines after the peptides have reached the periplasm. If cytoplasmicexpression of antibodies is preferred, said antibodies usually can beobtained at high yields from inclusion bodies, which can be easilyseparated from other cellular fragments and protein. In this case theinclusion bodies are solubilized in a denaturing agent such as e.g.guaridine hydrochloride (GndHCl) and then refolded by renaturationprocedures well known to those skilled in the art.

Plasmids expressing the scFv or Fab polypeptides are introduced into asuitable host, preferably a bacterial, yeast or mammalian cell, mostpreferably a suitable E. coli strain as for example JM83 for periplasmicexpression or BL21 for expression in inclusion bodies. The polypeptidecan be harvested either from the periplasm or form inclusion bodies andpurified using standard techniques such as ion exchange chromatography,reversed phase chromatography, affinity chromatography and/or gelfiltration known to the person skilled in the art.

The antibodies or antibody derivatives of the present invention can becharacterized with respect to yield, solubility and stability in vitro.Binding capacities towards TNF, preferably towards human TNFα, can betested in vitro by ELISA or surface plasmon resonance (BIACore), usingrecombinant human TNF as described in WO9729131, the latter method alsoallowing to determine the k_(off) rate constant, which should preferablybe less than 10⁻³s⁻¹. K_(d) values of <10 nM are preferred.

Aside from antibodies with strong binding affinity for human TNF, it isalso desirable to generate anti-TNF antibodies which have beneficialproperties from a therapeutic perspective. For example, the antibody maybe one which shows neutralizing activity in a L929 TNFalpha-mediatedcytotoxicity assay. In this assay toxicity of mouse L929 fibroblastcells treated with Actinomycin was induced with recombinant human TNF(hTNF). 90% of maximal hTNF-induced cytoxicity was determined to be at aTNF concentration of 1000 pg/ml.

All L929 cells were cultured in RPMI 1640 with phenolred, withL-Glutamine medium supplemented with fetal calf serum (10% v/v). Theneutralizing activity of anti-TNFα binders was assessed in RPMI 1640without phenolred and 5% fetal calf serum. Different concentrations(0-374 ng/mL) of anti-TNF binders are added to L929 cells in presence of1000 pg/ml hTNF in order to determine the concentration at which theantagonistic effect reaches half-maximal inhibition (EC50%) The doseresponse curve was fitted with nonlinear sigmoidal regression withvariable slope and the EC50 was calculated.

Optimized Variants

The antibodies of the invention may be further optimized for enhancedfunctional properties, e.g., for enhanced solubility and/or stability.

In certain embodiments, the antibodies of the invention are optimizedaccording to the “functional consensus” methodology disclosed in PCTApplication Serial No. PCT/EP2008/001958, entitled “Sequence BasedEngineering and Optimization of Single Chain Antibodies”, filed on Mar.12, 2008, which is incorporated herein by reference.

For example, the TNFα immunobinders of the invention can be comparedwith a database of functionally-selected scFvs to identify amino acidresidue positions that are either more or less tolerant of variabilitythan the corresponding position(s) in the VEGF immunobinder, therebyindicating that such identified residue position(s) may be suitable forengineering to improve functionality such as stability and/orsolubility.

Exemplary framework positions for substitution are described in PCTApplication No. PCT/CH2008/000285, entitled “Methods of ModifyingAntibodies, and Modified Antibodies with Improved FunctionalProperties”, filed on Jun. 25, 2008, and PCT

Application No. PCT/CH2008/000284, entitled “Sequence Based Engineeringand Optimization of Single Chain Antibodies”, filed on Jun. 25, 2008.For example, one or more of the following substitutions may beintroduced at an amino acid position (AHo numbering is referenced foreach of the amino acid position listed below) in the heavy chainvariable region of an immunobinder of the invention:

-   -   (a) Q or E at amino acid position 1;    -   (b) Q or E at amino acid position 6;    -   (c) T, S or A at amino acid position 7, more preferably T or A,        even more preferably T;    -   (d) A, T, P, V or D, more preferably T, P, V or D, at amino acid        position 10,    -   (e) L or V, more preferably L, at amino acid position 12,    -   (f) V, R, Q, M or K, more preferably V, R, Q or M at amino acid        position 13;    -   (g) R, M, E, Q or K, more preferably R, M, E or Q, even more        preferably R or E, at amino acid position 14;    -   (h) L or V, more preferably L, at amino acid position 19;    -   (i) R, T, K or N, more preferably R, T or N, even more        preferably N, at amino acid position 20;    -   (j) I, F, L or V, more preferably I, F or L, even more        preferably I or L, at amino acid position 21;    -   (k) R or K, more preferably K, at amino acid position 45;    -   (l) T, P, V, A or R, more preferably T, P, V or R, even more        preferably R, at amino acid position 47;    -   (m) K, Q, H or E, more preferably K, H or E, even more        preferably K, at amino acid position 50;    -   (n) M or I, more preferably I, at amino acid position 55;    -   (o) K or R, more preferably K, at amino acid position 77;    -   (p) A, V, L or I, more preferably A, L or I, even more        preferably A, at amino acid position 78;    -   (q) E, R, T or A, more preferably E, T or A, even more        preferably E, at amino acid position 82;    -   (r) T, S, I or L, more preferably T, S or L, even more        preferably T, at amino acid position 86;    -   (s) D, S, N or G, more preferably D, N or G, even more        preferably N, at amino acid position 87;    -   (t) A, V, L or F, more preferably A, V or F, even more        preferably V, at amino acid position 89;    -   (u) F, S, H, D or Y, more preferably F, S, H or D, at amino acid        position 90;    -   (v) D, Q or E, more preferably D or Q, even more preferably D,        at amino acid position 92;    -   (w) G, N, T or S, more preferably G, N or T, even more        preferably G, at amino acid position 95;    -   (x) T, A, P, F or S, more preferably T, A, P or F, even more        preferably F, at amino acid position 98;    -   (y) R, Q, V, I, M, F, or L, more preferably R, Q, I, M, F or L,        even more preferably Y, even more preferably L, at amino acid        position 103; and    -   (z) N, S or A, more preferably N or S, even more preferably N,        at amino acid position 107.

Additionally or alternatively, one or more of the followingsubstitutions can be introduced into the light chain variable region ofan immunobinder of the invention:

-   -   (aa) Q, D, L, E, S, or I, more preferably L, E, S or I, even        more preferably L or E, at amino acid position 1;    -   (bb) S, A, Y, I, P or T, more preferably A, Y, I, P or T, even        more preferably P or T at amino acid position 2;    -   (cc) Q, V, T or I, more preferably V, T or I, even more        preferably V or T, at amino acid position 3;    -   (dd) V, L, I or M, more preferably V or L, at amino acid        position 4;    -   (ee) S, E or P, more preferably S or E, even more preferably S,        at amino acid position 7;    -   (ff) T or I, more preferably I, at amino acid position 10;    -   (gg) A or V, more preferably A, at amino acid position 11;    -   (hh) S or Y, more preferably Y, at amino acid position 12;    -   (ii) T, S or A, more preferably T or S, even more preferably T,        at amino acid position 14;    -   (jj) S or R, more preferably S, at amino acid position 18;    -   (kk) T or R, more preferably R, at amino acid position 20;    -   (ll) R or Q, more preferably Q, at amino acid position 24;    -   (mm) H or Q, more preferably H, at amino acid position 46;    -   (nn) K, R or I, more preferably R or I, even more preferably R,        at amino acid position 47;    -   (oo) R, Q, K, E, T, or M, more preferably Q, K, E, T or M, at        amino acid position 50;    -   (pp) K, T, S, N, Q or P, more preferably T, S, N, Q or P, at        amino acid position 53;    -   (qq) I or M, more preferably M, at amino acid position 56;    -   (rr) H, S, F or Y, more preferably H, S or F, at amino acid        position 57;    -   (ss) I, V or T, more preferably V or T, R, even more preferably        T, at amino acid position 74;    -   (tt) R, Q or K, more preferably R or Q, even more preferably R,        at amino acid position 82;    -   (uu) L or F, more preferably F, at amino acid position 91;    -   (vv) G, D, T or A, more preferably G, D or T, even more        preferably T, at amino acid position 92;    -   (xx) S or N, more preferably N, at amino acid position 94;    -   (yy) F, Y or S, more preferably Y or S, even more preferably S,        at amino acid position 101; and    -   (zz) D, F, H, E, L, A, T, V, S, G or I, more preferably H, E, L,        A, T, V, S,

G or I, even more preferably A or V, at amino acid position 103. Inother embodiments, the immunobinders of the invention comprise one ormore of the solubility and/or stability enhancing mutations described inU.S. Provisional Application Ser. No. 61/075,692, entitled “SolubilityOptimization of Immunobinders,” filed on Jun. 25, 2008. In certainpreferred embodiments, the immunobinder comprises a solubility enhancingmutation at an amino acid position selected from the group of heavychain amino acid positions consisting of 12, 103 and 144 (AHo Numberingconvention). In one preferred embodiment, the immunobinder comprises oneor more substitutions selected from the group consisting of: (a) Serine(S) at heavy chain amino acid position 12; (b) Serine (S) or Threonine(T) at heavy chain amino acid position 103; and (c) Serine (S) orThreonine (T) at heavy chain amino acid position 144. In anotherembodiment, the immunobinder comprises the following substitutions: (a)Serine (S) at heavy chain amino acid position 12; (b) Serine (S) orThreonine (T) at heavy chain amino acid position 103; and (c) Serine (S)or Threonine (T) at heavy chain amino acid position 144.

As mentioned above, combinations of the VL and VH sequences, inparticular of those having the same or essentially the same set of CDRsequences but different framework sequences e.g. due to the presence ofthe substitutions mentioned above, can be shuffled and combined by alinker sequence. Exemplary combinations, without being limited to,include:

SEQ ID NO: 94 EP43 minEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSSGAMSWVRQAPGKGLEWVSVIISSGATYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 95 EP43maxEIVMTQSPSTLSASVGDRVIIKCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGFPSRFSGSGSGAEFTLTISGLEPADFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 96 EP43minmaxEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 97 EP43max DHPEIVMTQSPSTLSASVGDRVIIKCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGFPSRFSGSGSGAEFTLTISGLEPADFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGGPDDSNSMGTFDPWGQGTSVTVSS SEQ ID NO: 98 EP43minmaxDHPEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGGPDDSNSMGTFDPWGQGTSVTVSS SEQ ID NO: 99 EP43minmax VL: T22KEIVMTQSPSTLSASVGDRVIIKCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 100 EP43minmax: VL: V58FEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGFPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 101 EP43minmax VL: D81AEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPADFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 102 EP43minmax VL: Q79EEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLEPDDFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTLVTVSS SEQ ID NO: 103 EP1minEIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLAWYQQKPGKAPKLLIYDASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGYYRSGSGTANGSFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNDAISWVRQAPGKGLEWVSYISDWSIRYYANWAQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAPGAGDNGIWGQGTLVTVSS SEQ ID NO: 104 EP1maxEIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLAWYQQKPGKAPKLLIYDASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGYYRSGSGTANGSFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGIDLSNDAISWVRQAPGKGLEWVAYISDWSIRYYANWAQGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGAPGAGDNGIWGQGTTVTVSS SEQ ID NO: 105 EP1minmaxEIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLAWYQQKPGKAPKLLIYDASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGYYRSGSGTANGSFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGIDLSNDAISWVRQAPGKGLEWVAYISDWSIRYYANWAQGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGAPGAGDNGIWGQGTTVTVSS SEQ ID NO: 106 EP6minEIVMTQSPSTLSASVGDRVIITCQASESIYSGLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGFGTSNVENPFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSRYGVSWVRQAPGKGLEWVSTIGEAGRAYYANWARSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGEVFNNGWGAFNIWGQGTLVTVSS SEQ ID NO: 107 EP6maxEIVMTQSPSTLSASVGDRVIITCQASESIYSGLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTDFTLAISSLQPDDFATYYCQQGFGTSNVENPFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSRYGVSWVRQAPGKGLEWVGTIGEAGRAYYANWARSRSTISRDTSKNTVYLQMNSLRAEDTAVYYCARGEVFNNGWG AFNIWGQGTLVTVSSSEQ ID NO: 108 EP6minmaxEIVMTQSPSTLSASVGDRVIITCQASESIYSGLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGFGTSNVENPFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSRYGVSWVRQAPGKGLEWVGTIGEAGRAYYANWARSRSTISRDTSKNTVYLQMNSLRAEDTAVYYCARGEVFNNGWGAFNIWGQGTLVTVSS SEQ ID NO: 109 EP15minEIVMTQSPSTLSASVGDRVIITCQASENIYTSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGFATSNVENPFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGVSWVRQAPGKGLEWVSAIGETGRAYYANWAKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGEEFNNGWGAFNIWGQGTLVTVSS SEQ ID NO: 110 EP15maxEIVMTQSPSTLSASVGDRVIITCQASENIYTSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGFATSNVENPFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGFSLSRYGVSWVRQAPGKGLEWVGAIGETGRAYYANWAKSRSTISRDTSKNTVYLQMNSLRAEDTATYYCARGEEFNNGWGAFNIWGQGTTVTVSS SEQ ID NO: 111 EP15minmaxEIVMTQSPSTLSASVGDRVIITCQASENIYTSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGFATSNVENPFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGFSLSRYGVSWVRQAPGKGLEWVGAIGETGRAYYANWAKSRSTISRDTSKNTVYLQMNSLRAEDTATYYCARGEEFNNGWGAFNIWGQGTTVTVSS SEQ ID NO: 112 EP19minmodEIVMTQSPSTLSASVGDRVIITCQASDNIYRGLAWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCLGVYGYSSDDGAAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLNSNEISWVRQAPGKGLEWVSYIGNGGMTHYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSVEYTDLYYLNIWGQGTLVTVSS SEQ ID NO: 113 EP19maxmodEIVMTQSPSTLSASVGDRVIITCQASDNIYRGLAWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCLGVYGYSSDDGAAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLNSNEISWVRQAPGKGLEWVGYIGNGGMTHYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCASSVEYTDLYYLNIWGQGTLVTVSS SEQ ID NO: 114 EP34minEIVMTQSPSTLSASVGDRVIITCQSSQSVYGNIWMAWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISRSYWICWVRQAPGKGLEWVSCIYGDNDITPLYANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLGYADYAYDLWGQGTLVTVSS SEQ ID NO: 115 EP34maxEIVMTQSPSTLSASLGDRVIITCQSSQSVYGNIWMAWYQQKSGKAPKLLIYQASKLASGVPSRFSGSGSGAEFSLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFTISRSYWICWVRQAPGKGLEWVACIYGDNDITPLYANWAKGRFPVSTDTSKNTVYLQMNSLRAEDTAVYYCARLGYADYAYDLWGQGTLVTVSS SEQ ID NO: 116 EP35minEIVMTQSPSTLSASVGDRVIITCQASQSISNLLAWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSHTNVDNTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSVGYWICWVRQAPGKGLEWVSCIDAGTSGGTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVSSNGYYFKLWGQGTLVTVSS SEQ ID NO: 117 EP35maxEIVMTQSPSTLSASVGDRVIITCQASQSISNLLAWYQQKPGKAPKLLIVAASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGWSHTNVDNTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFSFSVGYWICWVRQAPGKGLEWVACIDAGTSGGTYYATWAKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGVSSNGYYFKLWGQGTTVTVSS SEQ ID NO: 118 EP35minmaxEIVMTQSPSTLSASVGDRVIITCQASQSISNLLAWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQGWSHTNVDNTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFSFSVGYWICWVRQAPGKGLEWVACIDAGTSGGTYYATWAKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGVSSNGYYFKLWGQGTTVTVSS SEQ ID NO: 119 EP42minEIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLAWYQQKPGKAPKLLIYDASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGYYRSGFGTANGSFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRNDAISWVRQAPGKGLEWVSYISDWGIKYYASWVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAPGAGDNGIWGQGTLVTVSS SEQ ID NO: 120 EP42maxEIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLAWYQQKPGKAPKLLIYDASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGYYRSGFGTANGSFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGIDLRNDAISWVRQAPGKGLEWVSYISDWGIKYYASWVKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGAPGAGDNGIWGQGTTVTVSS SEQ ID NO: 121 EP42minmaxEIVMTQSPSTLSASVGDRVIITCQSTESVYKNNYLAWYQQKPGKAPKLLIYDASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGYYRSGFGTANGSFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTVSGIDLRNDAISWVRQAPGKGLEWVSYISDWGIKYYASWVKGRFTISKDTSKNTVYLQMNSLRAEDTATYYCARGAPGAGDNGIWGQGTTVTVSS

In a preferred embodiment, a sequence has at least 90% identity, morepreferably at least 95% identity and most preferably 100% identity toanyone of sequences SEQ ID No. 94-121.

Uses of Anti-TNF Antibodies

For therapeutic applications, the anti-TNF antibodies of the inventionare administered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form such as those discussed above, including thosethat may be administered to a human intravenously as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. Theantibodies also are suitably administered by intra tumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

The anti-TNF antibodies are useful in the treatment of TNF-mediateddiseases. Depending on the type and severity of the disease, about 1μg/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily or weekly dosage might range from about 1μg/kg to about 20 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is repeated until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays, including, for example, radiographictumor imaging.

According to another embodiment of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for those purposes, such as vascular endothelialgrowth factor (VEGF), an antibody capable of inhibiting or neutralizingthe angiogenic activity of acidic or basic fibroblast growth factor(FGF) or hepatocyte growth factor (HGF), an antibody capable ofinhibiting or neutralizing the coagulant activities of tissue factor,protein C, or protein S (see Esmon et al., PCT Patent Publication No. WO91/01753, published 21 Feb. 1991), an antibody capable of binding toHER2 receptor (see Hudziak et al., PCT Patent Publication No. WO89/06692, published 27 Jul. 1989), or one or more conventionaltherapeutic agents such as, for example, alkylating agents, folic acidantagonists, anti-metabolites of nucleic acid metabolism, antibiotics,pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides,amines, amino acids, triazol nucleosides, or corticosteroids. Such otheragents may be present in the composition being administered or may beadministered separately. Also, the antibody is suitably administeredserially or in combination with radiological treatments, whetherinvolving irradiation or administration of radioactive substances. Theantibodies of the invention may be used as affinity purification agents.In this process, the antibodies are immobilized on a solid phase such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody is contacted with a sample containing the TNFprotein (or fragment thereof) to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the TNF protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theTNF protein from the antibody.

Anti-TNF antibodies may also be useful in diagnostic assays for TNFprotein, e.g., detecting its expression in specific cells, tissues, orserum. Such diagnostic methods may be useful in cancer diagnosis.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S.The antibody can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for exampleand radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981). Examples ofenzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) .beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate(e.g., P-nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-.beta.-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980. Sometimes, the label is indirectly conjugatedwith the antibody. The skilled artisan will be aware of varioustechniques for achieving this. For example, the antibody can beconjugated with biotin and any of the three broad categories of labelsmentioned above can be conjugated with avidin, or vice versa. Biotinbinds selectively to avidin and thus, the label can be conjugated withthe antibody in this indirect manner. Alternatively, to achieve indirectconjugation of the label with the antibody, the antibody is conjugatedwith a small hapten (e.g., digoxin) and one of the different types oflabels mentioned above is conjugated with an anti-hapten antibody (e.g.,anti-digoxin antibody). Thus, indirect conjugation of the label with theantibody can be achieved.

In another embodiment of the invention, the anti-TNF antibody need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the TNF antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of TNF protein in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radio nuclide (such as .sup.¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

The antibody of the present invention can be provided in a kit, apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit will include substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides thedetectable chromophore or fluorophore). In addition, other additives maybe included such as stabilizers, buffers (e.g., a block buffer or lysisbuffer) and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will provide areagent solution having the appropriate concentration.

Pharmaceutical Preparations

In one aspect the invention provides pharmaceutical formulationscomprising anti-TNF antibodies for the treatment of TNF-mediateddiseases. The term “pharmaceutical formulation” refers to preparationswhich are in such form as to permit the biological activity of theantibody or antibody derivative to be unequivocally effective, and whichcontain no additional components which are toxic to the subjects towhich the formulation would be administered. “Pharmaceuticallyacceptable” excipients (vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

A “stable” formulation is one in which the antibody or antibodyderivative therein essentially retains its physical stability and/orchemical stability and/or biological activity upon storage. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. Preferably, the formulation is stable at room temperature (about30° C.) or at 40° C. for at least 1 month and/or stable at about 2-8° C.for at least 1 year for at least 2 years. Furthermore, the formulationis preferably stable following freezing (to, e.g., −70° C.) and thawingof the formulation.

An antibody or antibody derivative “retains its physical stability” in apharmaceutical formulation if it shows no signs of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

An antibody or antibody derivative “retains its chemical stability” in apharmaceutical formulation, if the chemical stability at a given time issuch that the protein is considered to still retain its biologicalactivity as defined below. Chemical stability can be assessed bydetecting and quantifying chemically altered forms of the protein.Chemical alteration may involve size modification (e.g. clipping) whichcan be evaluated using size exclusion chromatography, SDS-PAGE and/ormatrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI/TOF MS), for example. Other types of chemicalalteration include charge alteration (e.g. occurring as a result ofdeamidation) which can be evaluated by ion-exchange chromatography, forexample.

An antibody or antibody derivative “retains its biological activity” ina pharmaceutical formulation, if the biological activity of the antibodyat a given time is within about 10% (within the errors of the assay) ofthe biological activity exhibited at the time the pharmaceuticalformulation was prepared as determined in an antigen binding assay, forexample. Other “biological activity” assays for antibodies areelaborated herein below.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and non-reducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kD (e.g. in the range from about 120 to about 400 kD). A“reducing sugar” is one which contains a hemiacetal group that canreduce metal ions or react covalently with lysine and other amino groupsin proteins and a “non-reducing sugar” is one which does not have theseproperties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Non-reducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof. Where it is desired that the formulation is freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes theantibody in the formulation. Non-reducing sugars such as sucrose andtrehalose are the preferred polyols herein, with trehalose beingpreferred over sucrose, because of the superior solution stability oftrehalose.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention has a pH in the range from about 4.5 to about6.0; preferably from about 4.8 to about 5.5; and most preferably has apH of about 5.0. Examples of buffers that will control the pH in thisrange include acetate (e.g. sodium acetate), succinate (such as sodiumsuccinate), gluconate, histidine, citrate and other organic acidbuffers. Where a freeze-thaw stable formulation is desired, the bufferis preferably not phosphate.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an antibody or antibody derivativerefers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody or antibody derivativeis effective. A “disease/disorder” is any condition that would benefitfrom treatment with the antibody or antibody derivative. This includeschronic and acute disorders or diseases including those pathologicalconditions which predispose the mammal to the disorder in question.

A “preservative” is a compound which can be included in the formulationto essentially reduce bacterial action therein, thus facilitating theproduction of a multi-use formulation, for example. Examples ofpotential preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferredpreservative herein is benzyl alcohol.

The present invention also provides pharmaceutical compositionscomprising one or more antibodies or antibody derivative compounds,together with at least one physiologically acceptable carrier orexcipient. Pharmaceutical compositions may comprise, for example, one ormore of water, buffers (e.g., neutral buffered saline or phosphatebuffered saline), ethanol, mineral oil, vegetable oil,dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, adjuvants, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione and/or preservatives. As noted above, other activeingredients may (but need not) be included in the pharmaceuticalcompositions provided herein.

A carrier is a substance that may be associated with an antibody orantibody derivative prior to administration to a patient, often for thepurpose of controlling stability or bioavailability of the compound.Carriers for use within such formulations are generally biocompatible,and may also be biodegradable. Carriers include, for example, monovalentor multivalent molecules such as serum albumin (e.g., human or bovine),egg albumin, peptides, polylysine and polysaccharides such asaminodextran and polyamidoamines. Carriers also include solid supportmaterials such as beads and microparticles comprising, for example,polylactate polyglycolate, poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose or dextran. A carrier may bear the compounds ina variety of ways, including covalent bonding (either directly or via alinker group), noncovalent interaction or admixture.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal, rectalor parenteral administration. In certain embodiments, compositions in aform suitable for oral use are preferred. Such forms include, forexample, pills, tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs. Within yet other embodiments, compositions providedherein may be formulated as a lyophilizate. The term parenteral as usedherein includes subcutaneous, intradermal, intravascular (e.g.,intravenous), intramuscular, spinal, intracranial, intrathecal andintraperitoneal injection, as well as any similar injection or infusiontechnique.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and may contain one or more agents, such as sweeteningagents, flavoring agents, coloring agent, and preserving agents in orderto provide appealing and palatable preparations. Tablets contain theactive ingredient in admixture with physiologically acceptableexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example, inert diluents (e.g., calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate), granulating and disintegrating agents (e.g., corn starch oralginic acid), binding agents (e.g., starch, gelatin or acacia) andlubricating agents (e.g., magnesium stearate, stearic acid or talc). Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monosterate or glyceryl distearatemay be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent(e.g., calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium (e.g., peanut oil, liquid paraffin or olive oil). Aqueoussuspensions contain the antibody or antibody derivative in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients include suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions may also comprise one or more preservatives, for exampleethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, and one or more sweetening agents, such assucrose or saccharin. Syrups and elixirs may be formulated withsweetening agents, such as glycerol, propylene glycol, sorbitol, orsucrose. Such formulations may also comprise one or more demulcents,preservatives, flavoring agents, and/or coloring agents.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil (e.g., arachis oil, olive oil, sesame oil, or coconutoil) or in a mineral oil such as liquid paraffin. The oily suspensionsmay contain a thickening agent such as beeswax, hard paraffin, or cetylalcohol. Sweetening agents, such as those set forth above, and/orflavoring agents may be added to provide palatable oral preparations.Such suspensions may be preserved by the addition of an anti-oxidantsuch as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil (e.g., olive oil orarachis oil), a mineral oil (e.g., liquid paraffin), or a mixturethereof. Suitable emulsifying agents include naturally-occurring gums(e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides(e.g., soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol), anhydrides (e.g., sorbitan monoleate), andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).An emulsion may also comprise one or more sweetening and/or flavoringagents.

The pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension in which the modulator, depending onthe vehicle and concentration used, is either suspended or dissolved inthe vehicle. Such a composition may be formulated according to the knownart using suitable dispersing, wetting agents and/or suspending agentssuch as those mentioned above. Among the acceptable vehicles andsolvents that may be employed are water, 1,3-butanediol, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils may be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectible compositions, and adjuvants such aslocal anesthetics, preservatives and/or buffering agents can bedissolved in the vehicle.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of modulator following administration). Such formulations maygenerally be prepared using well known technology and administered by,for example, oral, rectal, or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofmodulator release. The amount of an antibody or antibody derivativecontained within a sustained release formulation depends upon, forexample, the site of implantation, the rate and expected duration ofrelease and the nature of the disease/disorder to be treated orprevented.

Antibody or antibody derivatives provided herein are generallyadministered in an amount that achieves a concentration in a body fluid(e.g., blood, plasma, serum, CSF, synovial fluid, lymph, cellularinterstitial fluid, tears or urine) that is sufficient to detectablybind to TNF and prevent or inhibit TNF-mediated diseases/disorders. Adose is considered to be effective if it results in a discerniblepatient benefit as described herein. Preferred systemic doses range fromabout 0.1 mg to about 140 mg per kilogram of body weight per day (about0.5 mg to about 7 g per patient per day), with oral doses generallybeing about 5-20 fold higher than intravenous doses. The amount ofantibody or antibody derivative that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Dosage unitforms will generally contain between from about 1 mg to about 500 mg ofan active ingredient.

Pharmaceutical compositions may be packaged for treating conditionsresponsive to an antibody or antibody derivative directed to TNF.Packaged pharmaceutical compositions may include a container holding aeffective amount of at least one antibody or antibody derivative asdescribed herein and instructions (e.g., labeling) indicating that thecontained composition is to be used for treating a disease/disorderresponsive to one antibody or antibody derivative followingadministration in the patient.

The antibodies or antibody derivatives of the present invention can alsobe chemically modified. Preferred modifying groups are polymers, forexample an optionally substituted straight or branched chain polyalkene,polyalkenylene, or polyoxyalkylene polymer or a branched or unbranchedpolysaccharide. Such effector group may increase the half-live of theantibody in vivo. Particular examples of synthetic polymers includeoptionally substituted straight or branched chain poly(ethyleneglycol)(PEG), poly(propyleneglycol), poly(vinylalcohol) or derivatives thereof.Particular naturally occurring polymers include lactose, amylose,dextran, glycogen or derivatives thereof. The size of the polymer may bevaried as desired, but will generally be in an average molecular weightrange from 500 Da to 50000 Da. For local application where the antibodyis designed to penetrate tissue, a preferred molecular weight of thepolymer is around 5000 Da. The polymer molecule can be attached to theantibody, in particular to the C-terminal end of the Fab fragment heavychain via a covalently linked hinge peptide as described in WO0194585.Regarding the attachment of PEG moieties, reference is made to“Poly(ethyleneglycol) Chemistry, Biotechnological and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York and“Bioconjugation

Protein Coupling Techniques for the Biomedical Sciences”, 1998, M. Aslamand A. Dent, Grove Publishers, New York.

After preparation of the antibody or antibody derivative of interest asdescribed above, the pharmaceutical formulation comprising it isprepared. The antibody to be formulated has not been subjected to priorlyophilization and the formulation of interest herein is an aqueousformulation. Preferably the antibody or antibody derivative in theformulation is an antibody fragment, such as an scFv. Thetherapeutically effective amount of antibody present in the formulationis determined by taking into account the desired dose volumes andmode(s) of administration, for example. From about 0.1 mg/ml to about 50mg/ml, preferably from about 0.5 mg/ml to about 25 mg/ml and mostpreferably from about 2 mg/ml to about 10 mg/ml is an exemplary antibodyconcentration in the formulation.

An aqueous formulation is prepared comprising the antibody or antibodyderivative in a pH-buffered solution The buffer of this invention has apH in the range from about 4.5 to about 6.0, preferably from about 4.8to about 5.5, and most preferably has a pH of about 5.0. Examples ofbuffers that will control the pH within this range include acetate (e.g.sodium acetate), succinate (such as sodium succinate), gluconate,histidine, citrate and other organic acid buffers. The bufferconcentration can be from about 1 mM to about 50 mM, preferably fromabout 5 mM to about 30 mM, depending, for example, on the buffer and thedesired isotonicity of the formulation. The preferred buffer is sodiumacetate (about 10 mM), pH 5.0.

A polyol, which acts as a tonicifier and may stabilize the antibody, isincluded in the formulation. In preferred embodiments, the formulationdoes not contain a tonicifying amount of a salt such as sodium chloride,as this may cause the antibody or antibody derivative to precipitateand/or may result in oxidation at low pH. In preferred embodiments, thepolyol is a non-reducing sugar, such as sucrose or trehalose. The polyolis added to the formulation in an amount which may vary with respect tothe desired isotonicity of the formulation. Preferably the aqueousformulation is isotonic, in which case suitable concentrations of thepolyol in the formulation are in the range from about 1% to about 15%w/v, preferably in the range from about 2% to about 10% w/v, forexample. However, hypertonic or hypotonic formulations may also besuitable. The amount of polyol added may also alter with respect to themolecular weight of the polyol. For example, a lower amount of amonosaccharide (e.g. mannitol) may be added, compared to a disaccharide(such as trehalose).

A surfactant is also added to the antibody or antibody derivativeformulation. Exemplary surfactants include nonionic surfactants such aspolysorbates (e.g. polysorbates 20, 80 etc) or poloxamers (e.g.poloxamer 188). The amount of surfactant added is such that it reducesaggregation of the formulated antibody/antibody derivative and/orminimizes the formation of particulates in the formulation and/orreduces adsorption. For example, the surfactant may be present in theformulation in an amount from about 0.001% to about 0.5%, preferablyfrom about 0.005% to about 0.2% and most preferably from about 0.01% toabout 0.1%.

In one embodiment, the formulation contains the above-identified agents(i.e. antibody or antibody derivative, buffer, polyol and surfactant)and is essentially free of one or more preservatives, such as benzylalcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In anotherembodiment, a preservative may be included in the formulation,particularly where the formulation is a multidose formulation. Theconcentration of preservative may be in the range from about 0.1% toabout 2%, most preferably from about 0.5% to about 1%. One or more otherpharmaceutically acceptable carriers, excipients or stabilizers such asthose described in Remington's Pharmaceutical Sciences 21st edition,Osol, A. Ed. (2006) may be included in the formulation provided thatthey do not adversely affect the desired characteristics of theformulation. Acceptable carriers, excipients or stabilizers arenon-toxic to recipients at the dosages and concentrations employed andinclude; additional buffering agents; co-solvents; antioxidantsincluding ascorbic acid and methionine; chelating agents such as EDTA;metal complexes (e.g. Zn-protein complexes); biodegradable polymers suchas polyesters; and/or salt-forming counterions such as sodium.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation.

The formulation is administered to a mammal in need of treatment withthe antibody, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. In preferred embodiments, the formulationis administered to the mammal by intravenous administration. For suchpurposes, the formulation may be injected using a syringe or via an IVline, for example.

The appropriate dosage (“therapeutically effective amount”) of theantibody will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, the type ofantibody used, and the discretion of the attending physician. Theantibody or antibody derivative is suitably administered to the patentat one time or over a series of treatments and may be administered tothe patent at any time from diagnosis onwards. The antibody or antibodyderivative may be administered as the sole treatment or in conjunctionwith other drugs or therapies useful in treating the condition inquestion.

As a general proposition, the therapeutically effective amount of theantibody or antibody derivative administered will be in the range ofabout 0.1 to about 50 mg/kg of patent body weight whether by one or moreadministrations, with the typical range of antibody used being about 0.3to about 20 mg/kg, more preferably about 0.3 to about 15 mg/kg,administered daily, for example. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the pharmaceuticalformulation of the present invention, preferably an aqueouspharmaceutical formulation, and optionally provides instructions for itsuse. Suitable containers include, for example, bottles, vials andsyringes. The container may be formed from a variety of materials suchas glass or plastic. An exemplary container is a 3-20 cc single useglass vial. Alternatively, for a multidose formulation, the containermay be 3-100 cc glass vial. The container holds the formulation and thelabel on, or associated with, the container may indicate directions foruse. The article of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

EXEMPLIFICATION

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference in their entireties.

Throughout the examples, the following materials and methods were usedunless otherwise stated.

General Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques of polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992).

Thermostability Measurements

Attenuated total reflectance Fourier transform IR (FTIR-ATR) spectrawere obtained for various single chains and follow up molecules usingthe FT-IR Bio-ATR cell in a Tensor Bruker. The molecules wereconcentrated up to 3 mg/ml and dialyzed overnight at 4° C. against PBS,pH 6.5 and the buffer flow through was collected as blank. Thedenaturation profiles were obtained by thermo challenging the moleculeswith a broad range of temperatures in 5° C. steps (25 to 95° C.). Allspectra manipulations were performed using OPUS software. The mainbuffer and transient atmospheric (CO₂ and H₂O) background weresubtracted from the protein spectrum. The resulting protein spectrum wasthen baseline corrected and the protein amide I spectra was determinedfrom the width of the widest resolvable peak in the expected region.Second derivative spectra were obtained for the amide I band spectrausing a third degree polynomial function with a smoothing function.Changes in protein structure were estimated by amide I second derivativeanalysis using a linear calibration curve for the initial curve-fitcalculations assuming 0% denaturation for the 3 lower measurements and100% denaturation for the 3 higher measurements. The denaturationprofiles were used to approximate midpoints of the thermal unfoldingtransitions (TM) for every variant applying the Boltzmann sigmoidalmodel.

Solubility Measurements

Relative solubility of various scFv molecules was measured afterenhancing protein aggregation and precipitation in presence of ammoniumsulfate. Ammonium sulfate was added to the protein in aqueous solutionsto yield increments of 5% of saturation in the final mixturesalt-protein. The precipitation in the dynamic range was determinedempirically and the saturation intervals reduced in this range to 2.5%intervals saturation in the final mixture. After ammonium sulfateaddition, samples were gently mixed and centrifuged 30 minutes at 6000rpm. The remaining protein in supernatants was recovered for eachammonium sulfate percentage of saturation. Solubility curves weredetermined by measuring the protein concentration in the supernatantusing NanoDrop™ 1000 Spectrophotometer. Measurements of remainingsoluble protein in supernatants were normalized and used to estimatemidpoints of relative solubility for every variant applying theBoltzmann sigmoidal model.

Short Term Stability Test

Protein was examined after two weeks incubation at 40° C. for solubleaggregates and degradation products. Proteins with a concentration of 10mg/ml were dialyzed overnight at 4° C. against PBS with a broad range ofpHs (3.5, 4.5, 5.5, 6.5, 7.0, 7.5 and 8.5). Control protein with thesame concentration in standard buffer PBS (pH 6.5) was stored at −80° C.during the 2 weeks period. Determination of degradation bands bySDS-PAGE was done at t=0 and t=14 d time points and soluble aggregateswere assessed in the SEC-HPLC. Determination of remaining activity after2 weeks at 40° C. was done using Biacore.

Potency Assay

The neutralizing activity of anti-TNFa binders was assessed in a L929TNFa-mediated cytotoxicity assay. Toxicity of Mouse L929 fibroblastcells treated with Actinomycin was induced with recombinant human TNF(hTNF). 90% of maximal hTNF-induced cytoxicity was determined to be at aTNF concentration of 1000 pg/ml. All L929 cells were cultured in RPMI1640 with phenolred, with L-Glutamine medium supplemented with fetalcalf serum (10% v/v). The neutralizing activity of anti-TNFa-binders wasassessed in RPMI 1640 without phenolred and 5% fetal calf serum.Different concentrations (0-374 ng/mL) of anti-TNF binders are added toL929 cells in presence of 1000 pg/ml hTNF in order to determine theconcentration at which the antagonistic effect reaches half-maximalinhibition (EC50%) The dose response curve was fitted with nonlinearsigmoidal regression with variable slope and the EC50 was calculated.

Biacore binding analysis of anti-TNF scFvs

For binding affinity measurements at pH5 and pH 7.4 (data not shown),surface Plasmon resonance measurements with BIAcore™-T 100 were employedusing a NTA sensor chip and His-tagged TNF (produced at ESBATech). Thesurface of the NTA sensor chip consists of a carboxymethylated dextranmatrix pre-immobilized with nitrilotriacetic acid (NTA) for capture ofhistidine tagged molecules via Ni2+NTA chelation. Human TNFa N-histrimers (5 nM) are captured by the nickel via their N-terminal his-tagsand ESBA105 (analyte) is injected at several concentrations ranging from30 nM to 0.014 nM in 3 fold serial dilution steps. At the regenerationstep, the complex formed by nickel, ligand and analyte is washed away.This allows the use of the same regeneration conditions for differentsamples. The response signal is generated by surface Plasmon resonance(SPR) technology and measured in resonance units (RU). All themeasurements are performed at 25° C. Sensorgrams were generated for eachanti-TNF scFv sample after in-line reference cell correction followed bybuffer sample subtraction. The apparent dissociation rate constant(k_(d)), the apparent association rate constant (k_(a)) and the apparentdissociation equilibrium constant (K_(D)) were calculated usingone-to-one Langmuir binding model with BIAcore T100 evaluation Softwareversion 1.1.

Example 1 CDR Grafting and Functional Humanization of Monoclonal RabbitAnti-TNF Antibodies Grafting of Rabbit CDRs

Unlike traditional humanization methods which employ the human antibodyacceptor framework that shares the greatest sequence homology with thenon-human donor antibody, the rabbit CDRs were grafted into eitherframework FW1.4 (SEQ ID Nos. 1 and 2, linked by a (GGGGS)₄ linker (SEQID No: 72)) to generate a Min-graft or into the “rabbitized” frameworkrFW1.4 (SEQ ID No. 92) or its variant rFW1.4(v2) (SEQ ID No. 93) togenerate a Max-graft. Both frameworks were selected primarily fordesirable functional properties (solubility and stability), structuralsuitability to accommodate a large variety of rabbit CDRs and reasonablehomology to the rabbit variable domain consensus sequence. FrameworkrFW1.4 is a derivative of FW1.4 that was further engineered with the aimto serve as universal acceptor framework for virtually any set of rabbitCDRs. Although the stable and soluble framework sequence FW1.4 exhibitshigh homology to rabbit antibodies, it is not the most homologoussequence available.

Identification of Residues Potentially Involved in Binding

For each rabbit variable domain sequence, the nearest rabbit germlinecounterpart was identified. If the closest germline could not beestablished, the sequence was compared against the subgroup consensus orthe consensus of rabbit sequences with a high percentage of similarity.Rare framework residues were considered as possible result of somatichypermutation and therefore playing a role in antigen binding.Consequently, such residues were considered for grafting onto theacceptor framework rFW1.4 or rFW1.4(v2) to generate Max-grafts.Particularly, residues potentially implicated in direct antigen contactor influencing disposition of VL and VH were grafted. Further residuesdescribed to influence CDR structure were substituted if required. Noframework substitutions were made when CDRs were grafted onto FW1.4(Min-grafts). Examples of framework positions that were grafted toobtain the Max-grafts as disclosed herein can be identified by making asequence alignment of the framework regions of rFW1.4, rFW1.4(v2) andthe scFv sequences of interest provided herein. Webtools as known in theart may for example be used for said purpose (e.g. ClustalW orMultiAlin). All framework positions at which rFW1.4 and rFW1.4(v2)contain the same residue and at which the scFv of interest reveals adifferent residue, are framework positions that were grafted to obtainthe Max-grafts.

Domain Shuffling

Variable light chains of Min-grafts were combined with variable heavychain Max-grafts to identify optimal combinations in terms ofbiophysical properties (solubility and stability) and activity.

Cloning and Expression of scFvs

The scFvs described and characterized herein were produced as follows.The humanized VL sequences and the humanized VH sequences (SEQ IDNOs:51-88, without SEQ ID NO:72) were connected via the linker of SEQ IDNO:72 to yield an scFv of the following orientation:NH₂-VL-linker-VH-COOH (see e.g. SEQ ID NOs:94-121). In many cases DNAsequences encoding for the various scFvs were de novo synthesized at theservice provider Entelechon GmbH. The resulting DNA inserts were clonedinto the bacterial expression vector pGMP002 via NcoI and HindIIIrestriction sites introduced at the 5′ and 3′ end of the scFv DNAsequence, respectively. Between the DNA sequence of theVL domain and theVH domain, a BamHI restriction site is located. In some cases the scFvencoding DNA was not de novo synthesized, but the scFv expressingconstructs were cloned by domain shuffling. Accordingly, the VL domainswere excised and introduced into the new constructs via NcoI and BamHIrestriction sites, the VH domains via BamHI and HindIII restrictionsites. In other cases, point mutations were introduced into the VHand/or VL domain using state of the art assembling PCR methods. Thecloning of GMP002 is described in Example 1 of WO2008006235. Theproduction of the scFvs was done analogue as for ESBA105 as described inExamplel of WO2008006235.

Example 2 Profiling and Selection of Rabbit CDR Donor Antibodies

The general experimental procedure that was followed for the selectionof Rabbit antibodies (“RabMabs”) with TNF inhibitory activity Is asfollows: The rabbit antibodies were employed as donor antibodies forCDRs in the generation of highly soluble TNF immunobinders. Rabbits wereimmunized with TNFα prior to splenectomy. Splenocytes were isolated fromthe rabbits for the generation of hybridomas. A total of 44 hybridomaswere isolated and supernatants from these hybridomas were profiled forbinding affinity, biological potency, and binding specificity.

FIG. 1 depicts the relative ability of the supernatants from the 44anti-TNF RabMab hybridomas in neutralising TNFα in vivo. Neutralizationwas tested by measuring inhibition of cytotoxicity of TNFα in culturedmouse L929 fibroblasts. The supernatants display different efficacies inthe L929 assay. EC50 values (effective concentration to achieve 50%inhibition) were determined in a primary (blue bars) and secondaryscreens (red bars) and normalized with respect to the best performer ineach assay. TNF Binding affinity was also measured by BIACore analysisfor each RabMab (green bars). RabMabs encoded by each hybridoma werealso sequenced and the sequences subjected to phylogenetic analysisbased on the prediction of epitope clusters. Four representative Rabmabs(EPI-6, EPI-19, EPI-34, and EPI-43) with high binding activity andpotent neutralizing activity were selected from among differentphylogenetic families as donor antibodies for CDR grafting. Additionalfour (4) Rabmabs (EPI-1, EPI-15, EP-35 and EP-42) were selected for CDRgrafting based on their favorable activity in a secretion ELISA (seeFIG. 2).

Example 3 CDR Grafting and Functional Humanization of Rabbit DonorAntibodies

Unlike traditional humanization methods which employ the human antibodyacceptor framework that shares the greatest sequence homology with thenon-human donor antibody, the rabbit CDRs were grafted into a humanframework (FW 1.4) that was preselected for desirable functionalproperties (solubility and stability) using a Quality Control assay.Although the stable and soluble framework sequence exhibited highhomology with the RabMab, the selected acceptor antibody is not the mosthomologous sequence available.

A number of CDR grafts were generated for each of the rabmabs. The term“Min-graft” or “min” as used herein refers to a humanized variabledomain that was generated by grafting of rabbit CDRs from a rabbitvariable domain into a naturally occurring human acceptor framework (FW1.4, SEQ ID Nos. 1 and 2, linked by a (GGGGS)₄ linker (SEQ ID No: 72)).No changes in the framework regions are made. The framework itself waspreselected for desirable functional properties (solubility andstability). The term “Max-graft” or “max” as used herein refers to ahumanized variable domain that was generated by grafting of rabbit CDRsfrom a rabbit variable domain into the “rabbitized”, human acceptorframework “RabTor” (rFW1.4, SEQ ID No. 92), or into a derivative thereofreferred to as rFW1.4(v2) (SEQ ID No. 93). The “RabTor” framework wasprepared by incorporating conserved rabbit residues (otherwise which arerather variable in other species) at framework positions generallyinvolved in rabbit variable domain structure and stability, with the aimto generate a universally applicable framework that accepts virtuallyany set of rabbit CDRs without the need to graft donor frameworkresidues other than at positions that are different in their presumableprogenitor sequence, e.g. that were altered during somatic hypermutationand thus, possibly contribute to antigen binding. The presumableprogenitor sequence is defined to be the closest rabbit germlinecounterpart and in case the closest germline counterpart cannot beestablished, the rabbit subgroup consensus or the consensus of rabbitsequences with a high percentage of similarity. “Min-Max” or “minmax”refer to a humanized variable domain consisting of a “Min-graft”variable light chain combined with a “Max-graft” variable heavy chain,whereas “Max-Min” or “maxmin” refer to a humanized variable domainconsisting of a “Max-graft” variable light chain combined with a“Min-graft” variable heavy chain.

Table 2 shows a summary of the detailed characterization data forhumanized single chain antibodies that originate from eight differentmonoclonal rabbit antibodies or rabmabs (EP1, EP6, EP15, EP19, EP34,EP35, EP42 and EP43). So-called “min” grafts (e.g. EP1 min) refer toconstructs for which only the rabbit donor CDRs were grafted, whereasfor the so-called “max” grafts, not only the CDRs, but also some aminoacid positions in the donor framework were grafted. Additionally, thetable 2 shows the data for two His-tagged single-chain antibodies (EP34min C-His and EP19max C-His), as well as the reference single chainantibody ESBA105 described in WO 2006/131013. The third column, referredto as “L929” indicates the relative potencies of the different singlechain antibodies as determined in a L929 assay an compared to thepotency of ESBA105. The values for kon, koff and K_(D) are given inunits of M⁻¹s⁻¹, s⁻¹ and M, respectively. The seventh column gives themid point of thermally induced unfolding as determined with FT-IR. Thelast column indicates the relative yield of correctly folded proteinobtained from solubilized inclusion bodies after a refolding approach.

Some examples for BIACore data that went into table 2 are given in FIG.3: Binding kinetics for ESBA105 (FIG. 3 a), EP43max (FIG. 3 b) andEP34max (FIG. 3 c) binding to human TNFα are shown. Examples forcellular potency assays are given in FIG. 4, which compares ESBA105(closed circles) against EP43max (open squares) in a L929 assay. Furtherexamples of cellular potency assays that compare EP34max against themarketed antibodies infliximab and adalimumab are given in FIGS. 9 and10.

TABLE 2 Summary of the detailed characterization data for the fourrabbit monoclonals (EP6, EP19, EP34 and EP43) and their CDR graftedvariants. Description ID L929* kon koff K_(D) FT-IR TM° C. RF yield **EP1_min 1071 ND*** — — — — 2 EP6_min 673 ND*** 4.67E+04 4.94E−031.06E−07 50.2 35 EP15_min 1073 ND*** 1.57E+05 4.10E−02 2.62E−07 — 41.5EP19_min 616 ND*** — — — — — EP34_min 643 ND*** — — — — — EP35_min 1075ND*** — — — — 1 EP42_min 1076 ND*** 1.42E+05 8.35E−03 5.87E−08 — 3EP43_min 705 ND*** 5.38E+03 2.98E−02 5.54E−06 70.2 30.0 EP1_max 1072ND*** 1.11E+04 6.30E−04 5.69E−08 — 44 EP6_max 674 1.1 2.84E+05 1.45E−045.12E−10 48.1 12 EP15_max 1074  0.39 1.53E+06 2.26E−03 1.48E−09 68.657.8 EP19_max 1007 0.6 2.25E+04 6.54E−05 2.91E−09 53.5 52 EP34_max 79110.5  5.86E+05 1.68E−05 2.86E−11 72.4 4.05 EP35_max 1089  5.20 7.72E+051.50E−04 1.94E−10 — 0.66 EP42_max 1077 ND*** 1.21E+05 4.19E−04 3.46E−09— 47.6 EP43_max 676 6.4 1.78E+05 4.48E−05 2.51E−10 74.3 21.73EP34min_C-His 790 0.2 EP19max_C-His 789 1.9 *L929 [EC50-E105/EC50-X],compared in mass units [ng/ml] relative to the performance ofESBA105(WO06/131013) ** (mg/L refolding solution); ***Not Determined

Example 4 Solubility and Stability Optimization of EP43max, a PotentTNFα Binder

EP43max was selected for further optimization based on its potent TNFbinding activity. Biophysical characterization of this immunobinderrevealed that it exhibits a high midpoint of denaturation (Tm>70° C.) ina thermal unfolding assay (FTIR) (see FIG. 5). Nevertheless, EP43max wassubjected to solubility optimization to narrow its broad transitionphase in thermal unfolding. In order to improve the solubility of thenative EP43max, the three residue positions 12, 103, or 144 in the VHchain were substituted with amino acids with higher hydrophilicity. Thiscombination was shown to increase the solubility of the native proteinwithout affecting stability or binding activity. (VS at AHo position 12,V→T at AHo position 103, and L→T at AHo position 144) were introduced toreplace hydrophobic residues in the V-C domain interface of the variableheavy chain (VH) region of EP43max. In addition to the solubilityenhancing mutations, nine stabilizing mutations (T10S, K47R, Y57S, L91Fand T103V in the VL and E1Q, E6Q, S7T and V103L I the VH) wereidentified in EP43max (see Table 3). These stabilizing residues wereidentified from a functional consensus analysis of ESBATech's qualitycontrol (QC) frameworks. Stabilizing residues at positions 1 and 3 inthe VL and position 89 in the VH were already present in the EP43maxmolecule. An additional stabilizing mutation (M→L) was identified at VLposition 4, but was eliminated from consideration based on it predictedrole in antigen binding.

TABLE 3 Stabilizing Mutations in EP43max Parental Preferred StabilizingDomain Position Residue Substition Mutation VL 1 E E Already Present VL3 V V Already Present VL 4 M L Involved in Binding VL 10 T S T10S VL 47K R K47R VL 57 Y S Y57S VL 91 L F L91F VL 103 T V T103V VH 1 E Q E1Q VH6 E Q E6Q VH 7 S T S7T VH 89 V V Already Present VH 103 V L V103L Column1, Variable domain. Column 2, AHo amino acid position. Column 3,parental residue in EP43max. Column 4, preferred substitution for theposition indicated in column 2. Column 5, stabilizing mutation.

Example 5 Optimized Variants of EP43max, a Potent TNFα Binder

Tables 4 and 5 show the characterization data for three optimizedvariants of EP43max. EP43 maxDHP is solubility enhanced variant ofEP43max and comprises the three solubility enhancing mutations above (VSat AHo position 12, V→T at AHo position 103, and L→T at AHo position144). EP43_maxmin and EP43_minmax variants were generated by domainshuffling between “min” and “max” grafts. E.g., the “minmax” variantcomprises the minimal graft (CDR-graft only) version of the light chainand maximal graft version of the heavy chain (i.e., grafted rabbit CDRsplus rabbit framework residues involved in antigen binding) whereas, the“maxmin” variant comprised the maximal graft version of the light chainand the minimal graft version of the heavy chain.

TABLE 4 Characterization data for EP43max and variant thereof. FT-IRstability FW L929* Kon Koff KD TM° C. EP43_max 1.4 6.4 2.28E+05 5.68E−052.49E−10 74.32 EP43_maxDHP 1.4 6.7 2.35E+05 2.73E−05 1.16E−10 60.15EP43_maxmin 1.4 Inactive 1.46E+05 5.33E−03 3.66E−08 51.76 EP43_minmax1.4 1.6 2.28E+05 1.98E−04 8.68E−10 65.81 *L929 [EC50-E105/EC50-X],compared in mass units [ng/ml]

TABLE 5 Characterization data for EP43max and variant thereof. RefoldingRF yield Expression screening Purification EP43_max 27.73 +++ ok okEP43_maxDHP 17 +++ ok ok EP43_maxmin 11 +++ ok ok EP43_minmax 46 +++ okokThe thermal denaturation curves of EP43max and its optimized variantswere compared by FTIR analysis (see FIG. 6 and Table 6). EP43minmax wasfound to have a lower midpoint of unfolding than EP43max.

TABLE 6 Comparison of thermal denaturation curves of EP43max and itsoptimized variants by FTIR analysis EP43maxmin EP43max EP43minmaxEP43maxDHP (959) (676) (958) Tm° C. 60.15 65.81 77.78 51.76 Slope 2.6182.908 10.43 4.297 R² 0.9974 0.9969 0.9855 0.9936

sMoreover, the mimax variant exhibited a one-step unfolding transition,indicating that both domains unfold at very similar temperatures. EP43max (FIG. 7A) and its EP43minmax variant (FIG. 7B) were further comparedin a thermal stress test. Beta-sheet content and concentration ofsoluble protein were evaluated following thermal concentration atincreasing temperatures (50, 60 and 70° C.). EP43minmax was considerablymore stable than EP43max at the intermediate temperature of 60° C.

Example 6 Comparison of EP34max with Commercially Available TNFα Binders

The capacity of EP34max, Adalimumab and Infliximab to block cytotoxicactivity of 1000 pg/ml recombinant human TNFalpha was compared asdetailed above in a L929 assay. The capacity of EP43max, Adalimumab andInfliximab to block cytotoxic activity of 10 pg/ml recombinant humanTNFalpha was assessed in a Kym-1 assay. The results are shown in FIGS. 9a, b and FIGS. 10 a, b, respectively.

FIG. 9 a illustrates the potency of EP34max and Adalimumab to blockcytotoxic activity of 1000 pg/ml recombinant human TNFalpha (murine L929cells). The IC₅₀ for EP34max and Adalimumab was determined to be 1.03ng/ml and 8.45 ng/ml, respectively. FIG. 9 b illustrates the potency ofAdalimumab and EP34max to block cytotoxic activity of 10 pg/mlrecombinant human TNFalpha (human Kym-1 cells). The IC₅₀ for Adalimumaband EP34max (791) was determined to be 66.2 ng/ml and 0.69 ng/mlrespectively.

FIG. 10 a illustrates the potency of EP34max and Infliximab to blockcytotoxic activity of 1000 pg/ml recombinant human TNFalpha (murine L929cells). The IC₅₀ for EP34max and Infliximab was determined to be 1.04ng/ml and 13.9 ng/m, respectively. FIG. 10 b illustrates the potency ofInfliximab and EP34max (791) to block cytotoxic activity of 10 pg/mlrecombinant human TNFalpha (human Kym-1 cells). The IC₅₀ for Infliximaband EP34max was determined to be 14.98 ng/ml and 0.63 ng/mlrespectively. Thus, in both cases, EP34max showed better performance asInfliximab.

Other Embodiments

It is understood that the invention also includes any of themethodologies, references, and/or compositions set forth in the attachedAppendices A to E.

EQUIVALENTS

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations, web pages, figures and/or appendices, regardless of theformat of such literature and similar materials, are expresslyincorporated by reference in their entirety. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this application, including defined terms, term usage,described techniques, or the like, this application controls.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the present inventions have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present inventions encompass various alternatives, modifications,and equivalents, as will be appreciated by those of skill in the art.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made without departing fromthe scope of the appended claims. Therefore, all embodiments that comewithin the scope and spirit of the following claims and equivalentsthereto are claimed.

1. An immunobinder which specifically binds human TNFα, the immunobindercomprising: (a) a human heavy chain variable framework sequence and CDRH1, CDR H2 and CDR H3 sequences stemming from a rabbit immunobinder,wherein the human heavy chain variable region framework has at least 90%identity to SEQ ID NO:1; and (b) a human light chain variable frameworksequence and CDR L1, CDR L2 and CDR L3 sequences stemming from a rabbitimmunobinder, wherein the human light chain variable region frameworksequence has at least 85% identity to SEQ ID NO:2.
 2. The immunobinderof claim 1, wherein the human heavy chain variable region framework isor comprises SEQ ID NO:1, SEQ ID NO: 89, or SEQ ID NO: 90, and the humanlight chain variable region framework sequence is or comprises SEQ IDNO: 2 or SEQ ID NO:
 91. 3. The immunobinder of claim 2, comprising oneor more substitutions in the heavy chain framework (VH) at a positionfrom the group consisting of positions H24, H25, H56, H82, H84, H89 andH108; and/or a substitution in the light chain framework (VL) atposition L87 according to the AHo numbering system.
 4. The immunobinderof claim 3, wherein the substitution is selected from the groupconsisting of threonine (T) at position H24, valine (V) at position H25,glycine (G) or alanine (A) at position H56, lysine (K) at position H82,threonine (T) at position H84, valine (V) at position H89 and arginine(R) at position H108 and threonine (T) at position L87 according to theAHo numbering system.
 5. The immunobinder of claim 2, whereinimmunobinder comprises a solubility enhancing substitution in at leastone of heavy chain amino positions 12, 103 and 144 (AHo numbering). 6.The immunobinder of claim 5, wherein the solubility enhancingsubstitution is selected from the group consisting of: (a) Serine (S) atposition 12; (b) Threonine (T) at position 103; and (c) Threonine (T) atposition
 144. 7. The immunobinder of claim 1, further comprising: (a)one or more CDR sequences being at least 80% identical to a sequenceselected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; (b) one or more CDRsequences being at least 80% identical to a sequence selected from thegroup consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; (c) one or more CDR sequencesbeing at least 80% identical to a sequence selected from the groupconsisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19 and SEQ ID NO: 20; (d) one or more CDR sequences beingat least 80% identical to a sequence selected from the group consistingof SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25 and SEQ ID NO: 26; (e) one or more CDR sequences being at least80% identical to a sequence selected from the group consisting of SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 andSEQ ID NO: 32; (f) one or more CDR sequences being at least 80%identical to a sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQID NO: 38; (g) one or more CDR sequences being at least 80% identical toa sequence selected from the group consisting of SEQ ID NO: 39, SEQ IDNO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44;or (h) one or more CDR sequences being at least 80% identical to asequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO:
 50. 8.The immunobinder of claim 7, wherein the one or more CDR sequences beingat least 80% identical to a sequence selected from the group consistingof SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7and SEQ ID NO: 8, and has a substitution in at least one of positions22, 74, 95, 97 and 99 of the light chain variable region (VL) accordingto the AHo numbering system.
 9. The immunobinder of claim 8, wherein thesubstitution is threonine (T) at position L22, phenylalanine (F) ortyrosine (Y) at position L74, glutamate (E) L95 or alanine (A) atposition L99, or a combination thereof.
 10. The immunobinder of claim 7,comprising: (a) a heavy chain variable region (VH) having at least 90%sequence identity to a sequence selected from the group consisting ofSEQ ID NO:51, SEQ ID NO:53 and SEQ ID NO: 55, and a light chain variableregion (VL) having at least 90% sequence identity to a sequence selectedfrom the group consisting of SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO: 56,SEQ ID NO: 57, SEQ ID NO:58 and SEQ ID NO: 59; (b) a heavy chainvariable region (VH) having at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:60 and SEQ ID NO:62,and/or a light chain variable region (VL) having at least 90% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:61 and SEQ ID NO:63; (c) comprising a heavy chain variable region(VH) having at least 90% sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:64 and SEQ ID NO:66, and/or a lightchain variable region (VL) having at least 90% sequence identity to asequence selected from the group consisting of SEQ ID NO:65 and SEQ IDNO:67; (d) a heavy chain variable region (VH) having at least 90%sequence identity to a sequence selected from the group consisting ofSEQ ID NO:64 and SEQ ID NO:66, and/or a light chain variable region (VL)having at least 90% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:65 and SEQ ID NO:67; (e) a heavy chainvariable region (VH) having at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:68 and SEQ ID NO:70,and/or a light chain variable region (VL) having at least 90% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:69 and SEQ ID NO:71; (f) a heavy chain variable region (VH) having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:73 and SEQ ID NO:75, and/or a light chainvariable region (VL) having at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:74 and SEQ ID NO:76; (g)a heavy chain variable region (VH) having at least 90% sequence identityto a sequence selected from the group consisting of SEQ ID NO:77 and SEQID NO:79, and/or a light chain variable region (VL) having at least 90%sequence identity to a sequence selected from the group consisting ofSEQ ID NO:78 and SEQ ID NO:80; (h) a heavy chain variable region (VH)having at least 90% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:81 and SEQ ID NO:83, and/or a light chainvariable region (VL) having at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:82 and SEQ ID NO:84; or(i) a heavy chain variable region (VH) having at least 90% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:85 and SEQ ID NO:87, and/or a light chain variable region (VL) havingat least 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:86 and SEQ ID NO:88.
 11. The immunobinder ofclaim 7, having at least 90% sequence identity to, preferably 100% toany of SEQ ID NO: 94 to SEQ ID NO:
 121. 12. The immunobinder of claim11, having at least 90% sequence identity to, preferably 100% to SEQ IDNO: 103, SEQ ID NO: 104 or SEQ ID NO:105, and also having a substitutionin at least one of positions 87, 89 and 92 of the light chain variableregion (VL) according to the AHo numbering system.
 13. The immunobinderof claim 12, having at least 90% sequence identity to, preferably 100%to any of SEQ ID NO: 21 to SEQ ID NO: 50, wherein the heavy chainframework has deletions at framework positions 85 and 88 according tothe AHo numbering system.
 14. The immunobinder of claim 13, having atleast 90% sequence identity to, preferably 100% to any of SEQ ID NO: 27to SEQ ID NO: 32, further having a substitution in at least one ofpositions 86 and 87 of the light chain variable region (VL) according tothe AHo numbering system, preferably threonine (T) at position L87 andglutamine (Q) at position L88.
 15. The immunobinder of claim 13, havingat least 90% sequence identity to, preferably 100% to any of SEQ ID NO:33 to SEQ ID NO: 38, further, having a substitution in at least one ofpositions 15, 48, 90 of the light chain variable region (VL) accordingto the AHo numbering system.
 16. The immunobinder of claim 13,comprising one or more CDR sequences being at least 80% identical to asequence selected from the group consisting of SEQ ID NO: 39 to SEQ IDNO: 44, having a substitution in at least one of positions 57 and 87 ofthe light chain variable region (VL) according to the AHo numberingsystem, preferably to valine (V) at position L57 and threonine (T) atposition L87.
 17. The immunobinder of claim 16, having a stabilityenhancing substitution in at least one of positions 1, 3, 4, 10, 47, 57,91 and 103 of the light chain variable region according to the AHonumbering system, preferably, a stability enhancing substitutionselected from the group consisting of (a) glutamic acid (E) at position1, (b) valine (V) at position 3, (c) leucine (L) at position 4; (d)Serine (S) at position 10; (e) Arginine (R) at position 47; (e) Serine(S) at position 57; (f) phenylalanine (F) at position 91; and (g) Valine(V) at position
 103. 18. The immunobinder of claim 7, comprising one ormore CDR sequences being at least 80% identical to a sequence selectedfrom the group consisting of SEQ ID NO: 45 to SEQ ID NO: 50, havingdeletions at framework positions 85 and 88 according to the AHonumbering system and having a stability enhancing substitution in atleast one of positions 1, 3, 4, 10, 47, 57, 91 and 103 of the lightchain variable region according to the AHo numbering system, preferablya stability enhancing substitution selected from the group consisting of(a) glutamic acid (E) at position 1, (b) valine (V) at position 3, (c)leucine (L) at position 4; (d) Serine (S) at position 10; (e) Arginine(R) at position 47; (e) Serine (S) at position 57; (f) phenylalanine (F)at position 91; and (g) Valine (V) at position
 103. 19. The immunobinderof claim 7, which is an antibody, scFv, Fab or Dab.
 20. An immunobinderhaving CDRs that are different from any CDR of SEQ ID NOs: 3-50, thatcompetes for binding to human TNFα with the immunobinder of claim
 7. 21.An immunobinder having CDRs that are different from any CDR of SEQ IDNOs: 3-50, that binds the same epitope on human TNFα as the immunobinderof claim
 7. 22. A composition comprising the immunobinder of claim 7,and a pharmaceutically acceptable carrier.
 23. An isolated nucleic acidmolecule encoding a variable heavy (VH) chain region and/or variablelight (VL) chain region of claim
 7. 24. An expression vector comprisingthe nucleic acid molecule of claim
 23. 25. A host cell comprising theexpression vector of claim
 24. 26. A method of treating or preventing ahuman TNFα-mediated disease in a subject, comprising administering to asubject the antibody of claim 7 such that the subject is treated for theTNFα-mediated disease.
 27. The method of claim 26, wherein theTNFα-mediated disease is selected from the group consisting of chronicand/or autoimmune states of inflammation in general, immune mediatedinflammatory disorders in general, inflammatory CNS disease,inflammatory diseases affecting the eye, joint, skin, mucuous membranes,central nervous system, gastrointestinal tract, urinary tract or lung,states of uveitis in general, retinitis, HLA-B27+ uveitis, Behcet'sdisease, dry eye syndrome, glaucoma, Sjögren syndrome, diabetes mellitus(incl. diabetic neuropathy), insulin resistance, states of arthritis ingeneral, rheumatoid arthritis, osteoarthritis, reactive arthritis andReiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiplesclerosis, Guillain-Barre syndrome, myasthenia gravis, amyotrophiclateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidneydisease, cystitis, psoriasis (incl. psoriatic arthritis), hidradenitissuppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome(synovitis, acne, pustulosis, hyperostosis and osteitis), acne, Sweet'ssydrome, pemphigus, Crohn's disease (incl. extraintestinalmanifestastations), ulcerative colitis, asthma bronchiale,hypersensitivity pneumonitis, general allergies, allergic rhinitis,allergic sinusitis, chronic obstructive pulmonary disease (COPD), lungfibrosis, Wegener's granulomatosis, Kawasaki syndrome, Giant cellarteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graftversus host disease, host versus graft reactions, rejection episodesfollowing organ or bone marrow transplantation, sytemic and local statesof vasculitis in general, systemic and discoid lupus erythematodes,polymyositis and dermatomyositis, sclerodermia, pre-eclampsia, acute andchronic pancreatitis, viral hepatitis, alcoholic hepatitis, postsurgicalinflammation such as after eye surgery (e.g. cataract (eye lensreplacement) or glaucoma surgery), joint surgery (incl. arthroscopicsurgery), surgery at joint-related structures (e.g. ligaments), oraland/or dental surgery, minimally invasive cardiovascular procedures(e.g. PTCA, atherectomy, stent placement), laparoscopic and/orendoscopic intra-abdominal and gynecological procedures, endoscopicurological procedures (e.g. prostate surgery, ureteroscopy, cystoscopy,interstitial cystitis), or perioperative inflammation (prevention) ingeneral, Alzheimer disease, Parkinson's disease, Huntington's disease,Bell' palsy, Creutzfeld-Jakob disease. Cancer-related osteolysis,cancer-related inflammation, cancer-related pain, cancer-relatedcachexia, bone metastases, acute and chronic forms of pain, irrespectivewhether these are caused by central or peripheral effects of TNFα andwhether they are classified as inflammatory, nociceptive or neuropathicforms of pain, sciatica, low back pain, carpal tunnel syndrome, complexregional pain syndrome (CRPS), gout, postherpetic neuralgia,fibromyalgia, local pain states, chronic pain syndroms due to metastatictumor, dismenorrhea. Bacterial, viral or fungal sepsis, tuberculosis,AIDS, atherosclerosis, coronary artery disease, hypertension,dyslipidemia, heart insufficiency and chronic heart failure.
 28. Thecomposition of claim 22, formulated for topical, oral, nasal, rectal orparental administration.
 29. A method of treating a TNF α-mediateddisease in a mammal, in particular in a human, comprising administeringto a subject the composition of claim 22 by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes.